Experimental basis for the combined use of tobramycin and immune preparations for treating acute Pseudomonas aeruginosa infections

The possibility of using tobramycin (Tb) in combination with P. aeruginosa polyvalent corpuscular vaccine (PaPCV) or pyocyanosis hyperimmune plasma (PHP) for the treatment of P. aeruginosa sepsis was experimentally studied. The combined use of Tb and PHP, administered in amounts corresponding to ED50 of each preparation used separately, ensured the survival of 90% of the infected mice, and the injected of PaPCV with ED50 of the antibiotic ensured the survival of 73% of the experimental animals. The combined use of Tb and the two immuno-preparations (PaPCV and PHP) for the treatment of P. aeruginosa infection proved to be more effective than their separate administrations.     

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).     

Dynamics of the contamination of mice during the treatment of burn sepsis due to Pseudomonas aeruginosa using tobramycin alone or combined with active and passive immunization

An experimental study was made with a view to finding out the possible bacteriological advantages of the combined use of tobramycin (Tb) and P. aeruginosa corpuscular polyvalent vaccine (PaCPV) or P. aeruginosa hyperimmune plasma (PaHIP) in burn sepsis caused by P. aeruginosa. The use of the median therapeutic dose of Tb (2.5 mg/kg body weight per day), alone or in combination with immunopreparations, ensured the survival rate of the animals equal to 100%. The contamination of the body with P. aeruginosa after treatment with Tb and PaHIP or PaCPV was lower than after the administration of Tb alone, this phenomenon becoming manifest starting from day 5 of observation in the first case and from day 10 in the second case. The combined use of Tb and immunopreparations (PaCPV or PaHIP) in acute P. aeruginosa infection proved to be more effective than treatment with Tb alone.     

Use of a polyvalent erythrocyte diagnostic agent for determining antibodies to Pseudomonas aeruginosa in clinical practice

The use of polyvalent erythrocyte diagnosticum prepared on the basis of 5 polysaccharide antigens of P. aeruginosa slime, isolated from strains belonging to the most widespread serovars, makes it possible to check up the humoral response of donors after their immunization with P. aeruginosa polyvalent corpuscular vaccine with the aim of obtaining anti-P. aeruginosa donor plasma. Antibody titers, determined in the passive hemagglutination test with the use of the proposed diagnosticum and corresponding to a serum dilution of 1:320 and greater, age tentatively diagnostic, which may be indicative of P. aeruginosa in the development of purulent septic complications in patients. The use of the passive hemagglutination test with the newly developed polyvalent erythrocyte diagnosticum makes it possible to check up the specific response of patients having P. aeruginosa infection in the process of their treatment with anti-P. aeruginosa hyperimmune plasma used as a part of complex therapy.     

Morphological study of the organs and tissues of mice infected after a burn with a Pseudomonas aeruginosa strain and treated with a Pseudomonas aeruginosa polyvalent corpuscular vaccine and tobramycin

The effect of tobramycin and polyvalent corpuscular Ps. aeruginosa vaccine on the infectious process in mice with grade III burns inoculated with Ps. aeruginosa 1312 was studied. The highest percentage of the survival (100 per cent) among the animals was observed, when the vaccine was applied locally every day for 7 days. With the use of tobramycin administered intramuscularly for 2 times 95 per cent of the animals survived. When the vaccine was administered subcutaneously, 96.6 per cent of the animals survived. Morphological investigation of the organs and tissues of the mice showed that the vaccine applications to the infected burns promoted rapid elimination of microorganisms in the wounds and prevented development of sepsis due to Ps. aeruginosa. At the same time early epithelization of the wounds was observed (by the 4th-7th day). Intramuscular injections of tobramycin and subcutaneous injection of vaccine also prevented development of sepsis due to Ps. aeruginosa and protected the animals from death. Still no epithelization of the wounds by that period was observed. Microscopic examinations revealed necrosis of the epiderma and derma elements and microbial swarms on the skin surface.     

Effect of a corpuscular polyvalent Pseudomonas aeruginosa vaccine on the proliferation and differentiation of hematopoietic stem cells and erythropoiesis

P. aeruginosa corpuscular polyvalent vaccine stimulates hematopoiesis in sublethally stimulated mice. The stimulating effect is dose-dependent. The most effective method of immunization is the intravenous injection of the vaccine. The degree to which the stimulation of hematopoiesis is manifested varies in different strains of mice. The stimulation of hematopoiesis is not linked with an increase in the content of erythropoietin. P. aeruginosa polyvalent corpuscular vaccine and monovaccine, prepared from P. aeruginosa strain 1313 and incorporated into the polyvalent vaccine, protect lethally irradiated mice in the postradiation survival test.     

Effectiveness of tobramycin and immunologic preparations in experimental Pseudomonas infection

Certain indices of immunity were studied in mice with burn sepsis due to P. aeruginosa during their treatment with tobramycin (Tb) alone or in combination with immunological drugs. The most significant stimulation of the phagocytic function of peritoneal macrophages was observed when Tb was used in combination with polyvalent corpuscular vaccine of P. aeruginosa. When Tb was used alone or in combination with hyperimmune plasma of P. aeruginosa there was observed close correlation between the phagocytic index and the levels of cyclic adenosine-3',5'-monophosphate in them. Therapy of P. aeruginosa infection with the antibiotic and immunological drugs resulted in much higher levels of agglutinine antibodies in blood serum of the mice than the therapy with Tb alone.     

Production and experimental testing of the polyvalency of corpuscular vaccine for the prevention of Pseudomonas aeruginosa infections II. Experimental testing of the immunogenicity of polyvalent corpuscular Pseudomonas aeruginosa vaccine

Newly developed P. aeruginosa vaccine has been shown to be safe and apyrogenic for experimental animals. Immunization with the vaccine in a single injection of 0.5 ml has been found to ensure the protection of 80--98% of mice from lethal infection caused by virulent vaccine strains, with the exception of P. aeruginosa strain No. 1311, for 9 weeks. Immunity to P. aeruginosa strain No. 1311 develops only by day 56 after vaccination. No sharp correlation between the specific agglutinin level and the degree of protective effect induced by the immunization of animals with the polyvalent vaccine has been established. The vaccine has been shown to possess high immunogenicity in respect to clinical P. aeruginosa strains belonging to different serotypes (homo- and heterological vaccine strains).     

Safety and sensitizing properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine and its effect on hematological indices

The acute and chronic toxicity, influence on hematological characteristics and sensitizing properties of P. aeruginosa polyvalent corpuscular vaccine have been studied in experiments on 3 species of animals. The acute experiment has shown that the LD50 of the preparation contains not less than 7800 million cells, which is almost 160 times higher than the recommended immunizing dose (500 million cells). The safety of the preparation is confirmed by the data obtained in the histological and histochemical investigations of the tissues and organs of animals subjected to multiple immunizations with the vaccine. These investigations have revealed no pathological changes in the animals. During the study of the chronic toxicity of the preparation the hematological characteristics of the animals have been found to remain within normal limits. The vaccine has been shown to possess low sensitizing activity, which is manifested by the absence of severe reactions to allergic skin tests with different bacterial allergens (specific allergens obtained from P. aeruginosa and allergens obtained from other bacterial species), made on completion of the course of immunization with the vaccine.     

Isolation and study of the properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine. An evaluation of the biological properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine

P. aeruginosa killed polyvalent corpuscular vaccine, tested in a trial on 42 volunteer donors, is safe, low reactogenic and possesses pronounced immunogenicity, as the injection of the vaccine induced a rise in the titer of antibodies to P. aeruginosa up to 1:1280 in 95% of the immunized donors. To obtain specific antibodies in the blood plasma of the donors in an amount sufficient for the protective activity of the plasma becoming manifest, it is expedient to use the vaccination schedule providing for subcutaneous injections of 0.5, 0.5 and 1.0 ml at intervals of 7 days. Intense immunity thus induced in the donors lasts for 3-4 months. The hyperimmune plasma obtained from the donors has an antibody titer of at least 1:320 and shows a 90-100% protective effect in mice infected intraperitoneally with P. aeruginosa. The preliminary results of the clinical trial of anti-P. aeruginosa plasma have demonstrated its efficiency as a part of the complex treatment of patients with purulent septic complications of P. aeruginosa etiology.     

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.     

Results of using polyvalent Pseudomonas aeruginosa vaccine in children with burns by various medical centers

Polyvalent Pseudomonas aeruginosa vaccine, prepared at the Institute of Hematology from 10 hospital strains isolated from burn wounds, was administered to 32 children with extensive and deep burns. The vaccine was well tolerated. The vaccine produced a high degree of the immunity against Pseudomonas aeruginosa infection. Agglutinin serum titre increased significantly. Vaccination either prevented or inhibited the infection of burn wounds with Pseudomonas aeruginosa in all immunized children. The symptoms of Pseudomonas aeruginosa infection usually disappeared following one or two vaccinations. Bacteriemia caused by P. aeruginosa was not observed in 31 out of 32 children. In the remaining child transient bacteriemia was noted. No septicemia caused by P. aeruginosa was seen. Due to the high efficiency of the polyvalent P. aeruginosa vaccine all burned children with burns exceeding 10% of the total body surface should by vaccinated to prevent the life-threatening infections with Pseudomonas aeruginosa.     

Antibiotic sensitivity of different antigenic variants of Pseudomonas aeruginosa isolated in a hospital

The serological typing of 96% of P. aeruginosa strains isolated in hospitals has been carried out by means of agglutinating sera, and the tendency towards the prevalence of the strains of group V (25.3%) and serovar O11 (18.1%) has been revealed. The determination of the antibiotic sensitivity of different variants has shown that the strains of groups I, V and serovar O11 possess pronounced resistance to a wide spectrum of antibiotics, their resistance to gentamycin and polymixin being higher than in the strains of other serotypes. The determination of antibiotic resistance can serve only as an additional test in the serological typing of P. aeruginosa.     

Development of a polyvalent erythrocyte diagnostic agent based on the antigens of Pseudomonas aeruginosa slime

The study has revealed the fundamental possibility, and established the concrete conditions of obtaining standard erythrocyte polyvalent diagnosticum for the detection of antibodies to the antigens of P. aeruginosa, belonging to 5 types most frequently occurring in clinical practice, in the passive hemagglutination test. The data on high type- and species-specificity of the new erythrocyte diagnosticum have been obtained, which indicates that slime antigens have been correctly chosen as sensitins and confirms the necessity of using the polyvalent preparation. Preliminary data on the clinical trial of the erythrocyte diagnosticum show the expediency of its use for the control of specific immune response in donors in the process of obtaining anti-P. aeruginosa hyperimmune plasma, as well as the possibility of using this method for the serological diagnosis of P. aeruginosa infection. The diagnostic preparation has been found to retain its activity for 10 months (the term of observation).     

Principle of constructing polyvalent Pseudomonas aeruginosa vaccines]

Dependence of the range of protective action of P. aeruginosa vaccine on the number of its composites was studied. A principle of the selection of strains who vaccines differed in vivo by immunological specificity was applied to construction of the experimental preparations and modelling a polyvalent vaccine. Increase of the number of components in the vaccine was accompanied by increase of its protective action range. However, with the increase of the number of polyvaccine components in the polyvaccine the accretion of the protective effect expressed in the mean protective index per component displayed a gradual reduction. It was calculated theoretically that a 6--7-component vaccine should provide protection from 94--96% of the P. aeruginosa strains; as to further increase of the number of components--it would induce overloading of the vaccine with a possible absence of any effect.     

In vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia

Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia , but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na⁺ and K⁺, but not Cl⁻, are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.     

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).     

Antigenic complexes of Pseudomonas aeruginosa slime: their isolation and biological properties

The possibility of using antigenic complexes contained in the extracellular slime of P. aeruginosa clinical strains belonging to different serological groups as the components of a chemical vaccine has been revealed. Animal experiments have demonstrated a high immunogenicity of these preparations, as well as their low toxicity. The use of slime antigens stimulates the production of specific antibodies exerting a protective action against infection with homologous P. aeruginosa strains.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.