Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately 80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunoglobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studied in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD50 of five strains of P. aeruginosa was increased greater than or equal to 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MAbs in mice challenged with approximately 10 LD50 of the homologous LPS serotype ranged from less than 0.01 mg/kg to 1.00 mg/kg.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

Immunological study of antisera to to artificial O-antigen of Pseudomonas aeruginosa

O-sera were obtained by immunizing rabbits with artificial antigens: polysaccharide extracted from the lipopolysaccharide of P. aeruginosa (strain No. 868; 03a, 3d, 3e), or the high-molecular-weight or low-molecular-weight fraction of this polysaccharide, complexed with a natural protein (human IgG or rabbit globulin). The antisera to these antigenic complexes were highly O-specific. Antisera to the complexes of polysaccharide-protein and high-molecular-weight fraction-protein were more active in the passive haemagglutination reaction, slide agglutination test and immunodiffusion test in agar gel than was antiserum to the low-molecular-weight fraction-protein complex. The artificial antigens prepared and employed in the study are apt to be used for the preparation of monospecific immune sera.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

Obtaining antisera to Pseudomonas aeruginosa and Proteus antigens for performing immunoenzyme analyses in clinical practice

The data presented in this work indicate that specific antisera to P. aeruginosa and Proteus antigens can be produced by using extracts from these microorganisms, destroyed by ultrasonic treatment or by multiple freezing and thawing, for the immunization of rabbits. Blood serum samples from patients with purulent septic complications were studied for the presence of P. aeruginosa and Proteus antigens in ELISA with the use of peroxidase-labeled antibodies from antisera to P. aeruginosa and Proteus. This investigation revealed that during the first 3 days from the beginning of the clinical manifestations of the complications P. aeruginosa and Proteus antigens were detected in 86.4% and 83.4% of the patients, respectively. In the subsequent bacteriological study of wound discharge from these patients the corresponding microflora was detected.     

Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.     

Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.     

Surface haemagglutinating activity of Pseudomonas aeruginosa

Intact cells of several strains of Pseudomonas aeruginosa agglutinate papain-treated human erythrocytes. The agglutinating activity appears to reside in the surface layers of the bacterium-Pseudomonas surface haemagglutinin. This activity does not correlate with the existence of the internal PA-I and PA-II lectins, the presence of fimbriae or adherence to human buccal epithelial cells. Disruption of the bacterial cells by sonication abolishes their haemagglutinating activity. The intact cells of P. aeruginosa are also able to agglutinate rabbit, chicken, dog, guinea pig and sheep erythrocytes. This activity is generally higher with papain-treated erythrocytes, except those of rabbit in which lower haemagglutinating activity is observed after papain treatment. Optimal conditions for the haemagglutination are 37 degrees C and pH 6-7. Simple sugars do not inhibit, while fetuin and hydrophobic amino acids inhibit this activity. Exposure of the bacterial cells to proteolytic enzymes, EDTA or denaturating conditions abolish the haemagglutinating activity. These results indicate that the surface haemagglutinin is a protein which agglutinates red blood cells via hydrophobic interactions.     

Protective effect of a corpuscular polyvalent Pseudomonas aeruginosa vaccine in generalized chronic Pseudomonas aeruginosa infection in mice with cyclophosphamide-induced leukopenia

The prophylactic effect of immunization with P. aeruginosa polyvalent corpuscular vaccine has been shown on the model of P. aeruginosa generalized chronic infection in mice with leukopenia induced by the intraperitoneal injection of cyclophosphamids. This effect is manifested by the increased resistance of the animals to sublethal doses of P. aeruginosa strain, as well as by more intense general and specific immunological responses in the infected animals (the increase of specific antibody titers, the number of leukocytes in the blood serum and the phagocytic activity of the cells of peritoneal exudate).     

Gentamycin activity in combination with etonium in relation to Pseudomonas aeruginosa]

Gentamicin is one of the most effective drugs for treatment of infections caused by Ps. aeruginosa. However, isolation of the strains resistant to the antibiotics was not infrequent. It was shown in the experiments with 40 cultures that the activity of gentamicin against Ps. aeruginosa increased approximately 20 times when it was used in combination with ethonium, a derivative of bis-quaternary ammonium compounds.     

Investigations on the efficacy of monovalent Pseudomonas aeruginosa vaccine for oral administration in Pseudomonas aeruginosa experimental infection

Therapeutic efficacy of Pseudomonas aeruginosa vaccine for oral use (10(10) killed germs/ml), prepared from strain 4922, belonging to serotype XV, by Meitert-Meitert scheme, on 4 experimental models in mice (pneumonia, infected burn, septicaemia and urinary tract infection) was studied in comparison with monovalent Ps. aeruginosa vaccine serotype XV (10(9) killed germs/ml) for subcutaneous use and also with associated administration of the two vaccine variants. Mice immunization by using vaccine for oral use was performed by 0.5 ml vaccine per day, for 10 days and vaccine for subcutaneous use was administrated in a volume of 0.5 ml x 2, at 3 days interval. Mice immunization by using the two vaccine types, in association was concomitantly performed and in the same quantity as for separate immunization. In experimental pneumonia, Ps. aeruginosa vaccine for oral use protected mice in 35% of cases, those with infected burns were protected in 33.3% of cases, those with septicemia--in 96.6% of cases and those with urinary tract infection in 50% of cases. As compared to Ps. aeruginosa vaccine for subcutaneous use, the results obtained by vaccine for oral use are less favourable but associated administration of both vaccine variants led to superior results. Thus, in experimental pneumonia, it was obtained a surviving rate of 65% for animals immunized with both vaccine types, in comparison with 50% for animals immunized with vaccine for subcutaneous use only, and in Ps. aeruginosa infected burn, it was obtained a recovering rate of 79.1% for the animals immunized by using both vaccines, in comparison with 70.8% surviving for animals immunized with vaccine for subcutaneous use. In experimental septicaemia and urinary tract infection, combined use of both vaccine variants determined animals surviving and recovering in percents similar to those obtained by separate administration of vaccine for subcutaneous use (in septicemia--100% protection; in urinary tract infection--75% protection).     

Effectiveness of a polyvalent corpuscular Pseudomonas aeruginosa vaccine, antibiotics and their combinations in experimental chronic Pseudomonas aeruginosa infection

The data on the development of the experimental model of P. aeruginosa chronic infection in mice, produced by their intraperitoneal inoculation with the infective agent, and on the study of the properties of this model are presented. The model has been used in the experimental study of the preventive action of P. aeruginosa polyvalent corpuscular vaccine. The comparative study, carried out with the use of the proposed model, has been made with a view to evaluating the effectiveness of different methods for the treatment of P. aeruginosa chronic septic infection by means of antibiotics (polymixin B and tobramycin), P. aeruginosa polyvalent corpuscular vaccine and their combination. The combined use of this vaccine with antibiotics (polymixin B or tobramycin) has proved to give the most pronounced curative effect with respect to P. aeruginosa chronic infection.     

Single live cell imaging of chromosomes in chloramphenicol-induced filamentous Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading opportunistic pathogen in human infections, and it is renowned for its intrinsic resistance to structurally and functionally unrelated antibiotics. Filamentation induced by antibiotics appears to trigger bacteria to depart from a normal growth phase and enter a stationary growth phase. As antibiotic concentrations decline below a therapeutic range, filamentous bacteria begin to divide normally, leading to a more rapid regrowth of the bacteria. Furthermore, filamentous bacteria are associated with an increase in endotoxin release. Moreover, the immune system of a patient needs to cope with uncharacteristic filamentous bacteria. Thus, it is biologically and clinically significant to study and understand bacterial filamentation. In this study, we investigate the frequencies, conditions, and characteristics of a filamentous P. aeruginosa at single cell and single chromosome resolutions. Our results show that filamentous cells (elongated rods) contain multiple copies of the cell's chromosome. It appears that the unsuccessful segregation of replicated chromosomes in an individual cell accompanies the formation of undivided filamentous cells. The quantity of chromosomes and the length of the filamentous wild-type cells increase as the chloramphenicol concentration increases to 50 and 250 microg/mL, suggesting that chloramphenicol induces the filamentation. Filamentation in three strains of P. aeruginosa depends on the expression level of efflux pump (MexAB-OprM) and the minimum inhibitory concentration of chloramphenicol. This study also opens up the new possibility of real-time monitoring of modes of actions of antibiotics in live cells with both temporal and spatial resolution.     

Transfecting activity of Pseudomonas aeruginosa bacteriophage SM

Factors affecting the efficiency of transfection of Ps. aeruginosa PAO1 cells by the temperate SM bacteriophage DNA have been determined. The efficiency of transfection by DNA preparations isolated from the wild type bacteriophage SMc+ or its thermoinducible mutant SM cts6 is practically the same. The frequency of transfection is (7-9) X 10(4) of infectious centers per mkg of transfecting DNA. Variability in the frequencies of transfection has been registered depending of the infection conditions or on the transfer of the Ps. aeruginosa PAO1 recipient strain population into the competence phase. The efficiency of transfection is increased by the addition of Ca2+ or Mg2+ ions affecting the adsorption and absorbtion of phage DNA by the recipient cells. Optimal concentrations of the bivalent metal ions are 0.15M CaCl2 and 0.2M MgCl2. The results obtained have been used for optimizing the conditions of Ps. aeruginosa PAO1 transfection by SM bacteriophage DNA.     

Eicosanoid-mediated proinflammatory activity of Pseudomonas aeruginosa ExoU

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.     

铜绿假单胞菌体外抑菌活性研究

目的 观察铜绿假单胞菌抗菌物质对鲍曼不动杆菌等细菌的体外抑菌效果.方法 用交叉条带实验方法检测了铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌的体外抑制活性.结果 铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌体外抑菌活性良好,10株铜绿假单胞菌中,有8株对鲍曼不动杆菌的抑制率均达到了100％.另外有8株对耐甲氧西林表皮葡萄球菌的抑菌率均为100％;有6株对粪肠球菌的抑菌率为100％.结论 铜绿假单胞菌对上述3种致病菌具有较强的抗菌活性,具有开发前景.