Possible diagnosis of Pseudomonas aeruginosa infection by an immunoenzyme method using pyoimmunogen as the antigen

The possibility of detecting P. aeruginosa antibodies in patients by means of indirect solid-phase EIA techniques is shown. This assay is carried out with the use of reagents produced in the USSR: polystyrene assay plates manufactured by the Lenigrad Medpolymer Works are used as carriers, P. aeruginosa vaccine (pyoimmunogen) obtained under semi-industrial conditions at the Mechnikov Central Research Institute for Vaccines and Sera is used as antigenic complex and the commercial preparation produced by the Gamaleia Research Institute of Epidemiology and Microbiology serves as conjugate. The studies have revealed that in 95% of cases the level of antibodies in the sera of patients with acute destructive pneumonia accompanied by pleural empyema, abscesses of internal organs and acute hematogenic osteomyelitis is essentially higher than the level of "normal" antibodies in healthy donors from whom biologically confirmed P. aeruginosa cultures can be isolated. In the groups of patients with similar nosological forms of diseases caused by other infective agents such difference in antibody titers is not detected. These results suggest that the detection of antibodies to P. aeruginosa in patients' sera by means of EIA can be used as an additional test for the diagnosis of P. aeruginosa infections.     

Lipopolysaccharide identification in aqueous extracts of Pseudomonas aeruginosa by an immunoenzyme method

To determine the content of lipopolysaccharide (LPS) in P. aeruginosa aqueous extracts the ELISA was used. Membrane filtration and ultracentrifugation followed by precipitation with ammonium sulfate at 80% saturation and gel filtration on Sephadex G-100 have proved to be the most effective methods for purification of the aqueous extract from LPS.     

Production of cytotoxin by clinical strains of Pseudomonas aeruginosa

Presence of cytotoxin was studied in extracts of 57 strains of Pseudomonas aeruginosa (46 bacteremia, 4 environmental, and 7 Fisher immunotype), 10 Pseudomonas species, and 7 nonpseudomonas isolates. Cytotoxin was identified by Western immunoblot in extracts of all P. aeruginosa isolates. None of the Pseudomonas species or nonpseudomonas isolates were shown to produce this protein. No immunologic cross-reactivity was observed between cytotoxin antibody and P. aeruginosa alkaline protease, toxin A, or elastase. In partially purified extracts of two bacteremia strains and PA 158 (parent strain for cytotoxin production), detection of cytotoxin by Western immunoblot was correlated with biological activity, as measured by the cell swelling assay. Cytotoxin appears to be produced by all strains of P. aeruginosa and biological activity can be demonstrated in extracts of the strains tested. This biological activity is neutralized by specific antibody. Because of its known marked cytotoxic effect on most eukaryotic cells, P. aeruginosa cytotoxin might be an important factor in the pathogenesis of P. aeruginosa infections.     

Antiseptic sensitivity of clinical strains of Pseudomonas aeruginosa

MICs, the frequency of clinical and statistic resistance and the antiseptic activity index were studied in complex on out-of-hospital and hospital ecovars of P. aeruginosa. The forms resistant to a number of antiseptics, i.e. chloramine B, chlorhexidine, decamethoxine and dioxidine whose frequency eventually increased were shown to be widely distributed. The antiseptic sensitivity spectrum was more narrow and more heterogeneous than that of other bacteria, the heterogeneity level being dependent on the antiseptic type and bacterial ecovar. The activity of pervomur, phenol, resorcin and boric acid was higher against the clinical strains of P. aeruginosa while iodopyrin, sulfacetamide sodium and dioxidine were less active. The P. aeruginosa strains had natural resistance to cetylpyridinium chloride, rokkal, ethonium, sodium laurate and laurylsulfate and rivanol. It was recommended to assay antiseptic sensitivity of agents causing purulent inflammatory infections and to control circulation of antiseptic resistant variants of bacteria in hospitals.     

Pathogenicity of clinical strains of Pseudomonas aeruginosa]

The study of 208 Ps. aeruginosa clinical strains, introduced intraperitoneally into white mice, revealed the statistically significant prevalence of pathogenic cultures (87.5--100%). The pathogenic strains of Ps. aeruginosa were found to have statistically significant differences in their cultural and biochemical properties depending on the kind of clinical material: the strains isolated from blood formed mucoid colonies, the zones of hemolysis and thermolabile or thermostable alkaline phosphatase, and typing could be made in 100% of the isolated cultures; the strains isolated from material of closed cavities formed mucoid colonies and the zones of hemolysis; the pathogenic strains isolated from material of open cavities showed only a tendency towards greater activity in the formation of extracellular sline and greater capacity for the formation of clarification zones on yolk agar as compared with nonpathogenic cultures.     

Analysis of lectin binding by Bradyrhizobium japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin

A procedure was developed to assess the ability of wild-type and mutant strains of Bradyrhizobium japonicum to bind soybean lectin. The lectin-binding ability of bacteria grown on nitrocellulose filters was determined using peroxidase-labeled soybean lectin. The assay produced clear differences between strains known to be unable to bind soybean lectin and those which can. The assay gave results identical to those of the fluorescein isothiocyanate-soybean lectin-binding assay of T. V. Bhuvaneswari, S. G. Pueppke, and W. D. Bauer (1977, Plant Physiol. 60, 486-491) with regard both to the ability of particular B. japonicum strains to bind lectin and to the inhibition caused by N-acetyl-D-galactosamine. The method was used to screen Tn5-induced mutants of B. japonicum 2143 for their inability to bind soybean lectin. The procedure provides a sensitive and convenient method to screen Bradyrhizobium strains for the ability to bind soybean lectin.     

Virulence and toxigenicity of Pseudomonas aeruginosa cultures of various origins

The virulence and toxigenicity of newly isolated P. aeruginosa strains have been studied in experiments on white mice. These biological properties have been shown to be most pronounced in P. aeruginosa strains isolated from proteins, sometimes greatly exceeding those in strains isolated from healthy persons and the environment. Virulence and the factors which determine it are definitely interrelated in microorganisms and can vary, depending on the conditions of their habitat.     

Serotype characteristics of clinical strains of Pseudomonas aeruginosa]

A total of 348 P. aeroginosa strains isolated from patients with pulmonary and pleural diseases were studied, and 87% of the test showed the possibility of their serotyping with the use of group-specific agglutinating antisera. Serogroups II, III, IV were found to be prevalent among the strains isolated from patients with bronchopulmonary pathological states. Correlations between definite groups of P. aeroginosa in their sensitivity to antibiotics were established; thus, the cultures belonging the serogroups II, III, IV were found to be more sensitive to tetracycline annd chloramphenicol than the culture belonging to other serogroups.     

The estimation of usefulness of Pseudomonas bacteriophages in epidemiological investigations of Pseudomonas aeruginosa clinical strains

Out of 20 Pseudomonas phages, 17 were most suitable for typing of Pseudomonas aeruginosa strains isolated from different sources of human infections. These phages have been classified into three taxons based on coefficient of correlation of their lytic activity. Out of these strains only one appeared nontypeable. 240 distinquished phagotypes were classified into three groups and seven subgroups. This schema of classification was used in the epidemiological investigations of the Pseudomonas strains in relation to the category of infection and the place of isolation. Some statisticaly significant differences were detected. Various possibilities of applications of typing set of Pseudomonas phages are discussed.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Proteomic profiling of clinical and environmental strains of Pseudomonas aeruginosa

Molecular Biology Reports - Pseudomonas aeruginosa is a ubiquitous bacterium, which is able to change its physiological characteristics in response to different habitats. Environmental strains are...     

Antibiotic resistance study of clinical strains of Pseudomonas aeruginosa]

The study of 40 clinical strains of Ps. aeruginosa isolated from the wound surfaces of the patients showed that all the isolates were resistant to one or several antibiotics. The number of the strains resistant to 5, 4, 3, 2 or 1 drug was 5, 22.5, 25. 30 or 17.5 per cent respectively. Fifteen strains carried resistance plasmids capable of conjugative transfer. Eleven out of 21 plasmids controlled resistance to chloramphenicol, 7 plasmids controlled resistance to streptomycin and sulfanylamides, 1 plasmid controlled resistance to streptomycin and chloramphenicol. The presence of two types of the plasmids controlling resistance to chloramphenicol and streptomycin + sulfanylamides respectively was found. All the plasmids proved to be capable of conjugative transfer between the strains of Ps. aeruginosa ML (PAO). The frequency of the plasmid conjugative transfer in such crosses ranged from 10(-6) to 10(-3). Most of the plasmids belonged to the incompatibility groups P-2 and P-7. One plasmid belonged to the incompatibility group P-5. It should be noted that about a half of the plasmids (11 out of 21) belonged to the incompatibility group P-7 which up to the present time was conditional, since was represented by a single plasmid Rms 148.     

Fluoroquinolone susceptibility of Pseudomonas aeruginosa strains isolated from clinical materials

The goal of our research was an analysis of sensitivity of Pseudomonas aeruginosa to fluoroquinolones (SPX, PEF, UB, LOM, CIP, ENX, OFX, NOR). The sensitivity was tested by disk diffusion method, according to NCCLS standards. 120 strains isolated from hospitalized (76 strains) and outpatient clinic (44 strains) persons were tested. The highest sensitivity of strains was observed to norfloxacin (36.8% and 86.4% of strains, respectively) and ciprofloxacin (30.3% and 81.8%). None of tested microorganisms was sensitive to flumequine. Resistant strains, isolated from sick persons in outpatient clinics were fewer (13.6%) as compared to hospitalized persons (51.3%).     

Immunosuppressive components of extracellular polysaccharide from Pseudomonas aeruginosa clinical strains

The immunosuppressive activity of lipopolysaccharide (LPS) contained in filtrates of newly isolated P. aeruginosa cultures was studied. The experimental model of delayed hypersensitivity to non-bacterial antigen in CBA mice and gel filtration through Sephadex G-200 were used. In addition to the already known LPS component with direct immunosuppressive action and having a mol.wt. of 150-800 kD, a new component was detected. It was found to be in inactive state and could be activated by redox treatment, thus becoming capable of inducing capacity for immunosuppression in Escherichia coli. This component had a mol.wt. of 50-70 kD and lost its activity after heating.     

Serotyping of Pseudomonas aeruginosa strains by slide coagglutination

Serological typing of Pseudomonas aeruginosa strains (228 strains) by slide coagglutination, using our own reagents (5 polyvalent and 22 monovalent ones, corresponding to the 22 serotypes in Meitert-Meitert scheme), led to identical results obtained by conventional slide agglutination. Utilization of live Ps. aeruginosa cells suspensions, killed by boiling or autoclaving, showed a 100% concordance of results, when using the second and the third suspension types and a 97.37% one between them and the live cells suspension. We noticed that reactions intensity was higher when using bacterial suspensions, boiled for 2.5 hours, in comparison with autoclaved cells suspensions, 30 minutes at 120 C. Compared to conventional slide agglutination, the slide coagglutination presents more advantages, being simple, rapid, specific and economical.     

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.     

On the toxinogenicity of Pseudomonas aeruginosa strains

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.