Pseudomonas aeruginosa phage lysate as an immunobiological agent. 1. Selection of Pseudomonas aeruginosa clinical strains for phage lysate preparation.

A total of 2087 Pseudomonas aeruginosa isolates collected during the period 1994-1997 were used as starting material. Out of 1704 in-patient isolates, 299 strains were selected for the preparation of phage lysates but only five strains provided stable lysates, i.e., maintained the ability to be repeatedly and completely lysed by the appropriate phage in the course of several years. A set of 193 out-patients (189) and water sources (4) isolates failed to yield strains suitable for phage lysate preparation; 190 strains isolated abroad from patients with cystic fibrosis or respiratory infections included three isolates which, despite having a high degree of mucus production, were suitable for lysate preparation. The antigenic pattern of the phage lysates was ascertained by SDS-polyacrylamide gel electrophoresis.

A new bacteriophage typing scheme for Proteus mirabilis and Proteus vulgaris strains. 3. Analysis of lytic properties.

The lytic properties of 21 bacteriophages constituting a new typing set for Proteus were examined in 507 Proteus mirabilis and 29 P. vulgaris strains isolated from patients and healthy subjects. Comparison of their morphological, serological, genetic and lytic properties showed that, in the Myoviridae and Podoviridae families, some phages were so closely related that the presence of all of them in the set was redundant. Analysis of the lytic properties revealed that some of the bacteriophages were not active enough to facilitate the differentiation of Proteus strains. The size of the final typing set was reduced from 21 to 12 phages but it was suggested that, in order to improve the differentiation capacity of the set, new phages should be included.

A new phage typing scheme for Proteus mirabilis and Proteus vulgaris strains. 1. Morphological analysis.

A new bacteriophage typing set, composed of 22 phages, was established as a tool for differentiation of Proteus strains. All the phages were tailed and included 4 morphological types (A1, A2, B1 and C1). They were classified into the families Myoviridae, Siphoviridae and Podoviridae. From the set, 19 phages had double-stranded DNA and 3 were single-stranded DNA phages.

Comparison of the in vitro effects of several cephalosporins on the oxidative burst of human phagocytes.

The metabolic activity (oxygen radical formation) of human phagocytes was not substantially affected by the tested cephalosporins. Therapeutic concentrations caused only a mild suppression or immunopotentiation in some cases or there were no effects altogether.

Human neutrophil mobilization during open heart surgery.

Phagocyte released reactive oxygen species are often discussed in connection with ischemic and reperfusion injuries to the myocardium. The kinetics of the accumulation and oxidative burst of human blood phagocytes was studied by chemiluminescence during open heart surgery in the myocardium of human patients. Direct evidence is presented for an accumulation of neutrophils along with their markedly increased metabolic activity (oxygen radical formation), especially following the reperfusion of the ischemic myocardium. Leukocyte numbers and activity remained significantly elevated even in the venous blood obtained 24 h after the operation.

[A simple immunologic skin test in piglets]. / Jednoduchý imunologický kozní test u selat.

A simple immunological skin test was carried out on 73 weaned piglets at a weight of 15 to 30 kg. Bristles on the piglets' back were shaved off and 0.1-ml amounts of four substances were injected intradermally. These were an isotonic solution of sodium chloride (900 micrograms NaCl in 0.1 ml), histamine (100 mu g in 0.1 mu l), phytohaemagglutinin (200 mu g in 0.1 ml), and a staphylococcus lysate (STAVA). The sites of administration of the substances were 5 cm apart. The dermal reaction was evaluated 20 minutes, 24 and 48 h after the i.d. administration. The size of the papule (measured after 20 min) and the size of the induration (after 24 and 48 h) were measured in two perpendicular directions and the mean value was calculated from the measured data. The average size of the papule was 8.1 mm 20 min after administration of sodium chloride (NaCl) and 19.6 mm 20 min after administration of histamine (HIS). The average size of induration after the administration of phytohaemagglutinin (PHA) was 13.1 mm after 24 h and 11.0 mm after 48 h. The average size of the papule was 11.1 mm 20 min after the administration of STA, the average size of induration was 10.6 mm 24 h after administration of STA and 8.5 mm after 48 h. Piglets that had papule larger than 15 mm 20 min after HIS administration (after subtracting papule size after NaCl administration) were regarded as piglets susceptible to states of allergy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunologic skin tests in piglets]. / Imunologické kozní testy u selat.

Immunological skin tests were carried out in 213 weanling piglets at the weight of 5 to 30 kg. Immediate hypersensitivity was evaluated by help of diagnostic allergens (mould, yeast and bacterial ones), delayed hypersensitivity by help of staphylococcus lysate, cellular immunity (tests de novo) by help of phytohemagglutinin and as common recall antigens were used tuberculin, toxoplasmin, candidic and tetanic antigen. The above substances were applied intradermally to the back of the piglets. For an evaluation of non-specific inflammatory response, sodium lauryl sulphate applied epicutaneously was used. The average reaction to diagnostic allergens (50 PNU in 0.05 ml) evaluated after 20 minutes was characterized as light dermal reactions (the papule size of 3-5 mm); in 22% of piglets moderate dermal reactions (the papule larger than 6 mm) to the diagnostic bacterial allergen (Staphylococcus aureus, Staphylococcus epidermidis) were determined. After an intradermal implantation of histamine (50 micrograms in 0.05 ml) the average size of the papule was 13 mm in 20 minutes. After the application of 0.1 ml of staphylococcus lysate (STAVA), the induration exceeding 10 mm was observed in 13% of piglets in 24 hours. After the application of phytohemagglutinin (100 micrograms in 0.1 ml), the induration exceeding 5 mm in 24 hours was determined in 93% of piglets and in 48 hours in 59% of piglets. Common recall antigens were applied at the volume of 0.1 ml. Forty-eight hours after the application no palpable induration was determined: in 91% of piglets after the application of tuberculin (2 TU PPD in 0.1 ml); in 75% of piglets after toxoplasmin (according to PNY 30-33-74); in 98% of piglets after candidic antigen (100 PNU in 0.1 ml) and in 86% of piglets after tetanic antigen implantations (0.03 Lf in 0.1 ml). The epicutaneously applied sodium lauryl sulphate (at 2.5% and 5% concentrations) caused no inflammatory dermal reactions (erythema or induration) after 24 hours. In piglets it is best to apply phytohemagglutinin for evaluating cellular immunity, staphylococcus lysate for evaluating delayed hypersensitivity to staphylococci and histamine for obtaining the information on a disposition of piglets to allergic diseases.

Phage plaques and autoplaques in Pseudomonas aeruginosa strains.

Studies were made on the morphological variety in plaques produced by phage lysis and autoplaques in Pseudomonas aeruginosa strains. The great variability of phage plaque morphology may lead to a confusion with the autoplaques produced spontaneously by P. aeruginosa strains. The production of autoplaques is characteristic of a large number of clinical strains of P. aeruginosa. The appearance of autoplaques may complicate analysis of significant clinical strains of P. aeruginosa.

Further observations of Corynebacterium renale as an indicator organism in the detection of the exfoliation-positive strains of Staphylococcus aureus.

Exfoliatin-positive S. aureus strains of known phenotypes were examined for bacteriocin production using S. aureus 209P, C. pseudodiphtheriticum, or C. renale as indicator strains. Strains of the phenotype Bac+ Tox+ had a positive result when the simultaneous method was used, and produced a wide inhibition of the indicator strains, especially of C. renale, if the deferred method was used. The strains of the phenotype Bac- Tox+ were negative upon examination by the simultaneous method, but they caused narrow inhibition zones of the indicator strains to appear with the deferred method, especially with C. renale. S. aureus strains of diverse origin used as controls were in both methods negative with regard to inhibition of indicator strains. C. renale was found to be very suitable for the detection of bacteriocins of the exfoliatin-positive staphylococci, particularly when using the deferred method.

New possibilities of staphylococcin detection in the exfoliatin-producing strains of staphylococcus aureus.

C. renale was found to be highly sensitive to the effect of bacteriocin produced by the exfoliatin-positive strains of S. aureus. The sensitivity of other indicator organisms used was determined through descending series of C. pseudodiphtheriticum, C. diphtheriae, S. aureus strains Oxford 209P, UT 0017, and Wood 46. Strains of C. pyogenes only produced contradictory results which made any satisfactory interpretation impossible. A modification of the simultaneous bacteriocin-detection method of exfoliatin-positive S. aureus strains, which use suspension of an indicator strain in nutrient agar to obtain lawn homogeneity, has been employed. The use of the modified method together with C. renale to serve as the indicator strain represents a rapid and reliable test suitable for routine diagnostics.

Diagnostic utilization of hemolytically active exosubstances of certain Gram-positive bacteria. II. Detection of staphylococcal hemolysins by means of a pre-purified hemolytically active exosubstance of Corynebacterium pseudotuberculosis.

The use of Corynebacterium pseudotuberculosis or of its hemolytically active exosubstance for the detection of staphylococcal hemolysins represents a suitable alterative to the use of the beta-toxin producing strain of Staphylococcus aureus, or of isolated beta-toxin. An increased capture of beta-toxin production was recorded when the CAMP-factor of Streptococcus agalactiae was used. A total of 446 Staphylococcus aureus strains and 48 coagulase-negative staphylococal strains were tested against the prepurified form of hemolytically active exosubstance of Corynebacterium pseudotuberculosis and against the CAMP-factor. The results obtained are in agreement with the testing by means of staphylococcal beta-toxin and of the CAMP-factor. The two modifications of the method for the detection of staphylococcal hemolysins using one plate as proposed by the authors give good results. Their ability of assessment, however, is higher by one degree when the exocubstance of Corynebacterium pseudotuberculosis is employed.

A simple method of detecting staphylococcal hemolysins.

A modification of the "one-plate method" for detection of staphylococcal hemolysins has been described. Bacterial strains of Staphylococcus aureus, Streptococcus agalactiae and Cornebacterium pseudotuberculosis were replaced by their prepurified hemolytically active exosubstances: beta-toxin, CAMP-factor, and, the exosubstance of Corynebacterium pseudotuberculosis called COREX. By means of the modification described, exact detection of hemolysins in possible with staphylococcal strains producing one type of hemolysin as well as with strains producing a combination of hemolysins.

Therapeutic possibilities in Pseudomonas infections.

Acute pseudomonas infections require treatment with antibiotics producing a bactericidal effect. The most useful are gentamicin, tobramycin, sisomicin and polymyxin B. In resistant strains, amikacin is indicated in addition. Carbenicillin, ticarcillin, carfenicillin or azocillin should never be given alone but in combination with some of the above preparations. Other drugs, such as chloramphenicol, tetracycline or streptomycin, though effective in vitro, should be avoided. Chemotherapy may be complemented by passive immunization either with hyperimmune specific gama globulin or hyperimmune plasma. A programmatic item of combined treatment is active immunization, especially with toxoid vaccine. Chronic processes are not, perhaps with the exception of urinary infections, suitable for antibiotic therapy. For this reason effective polyvalent vaccines should be developed from appropriate strains. It is now certain that in infections caused by mucous strains (most frequently encountered in cystic fibrosis) the vaccine should be prepared from these strains, since they have distinct functional and antigenic characteristics.

Lytic manifestations in the bacterial strains of Pseudomonas aeruginosa (bacteriophages bacteriocines, autoplaques).

Ps. aeruginosa strains--a frequement resuet of an irresponsible antibiotic therapy--represent a common agent of nosocomial infektions. At the same time, gravity of Pseudomonas diseases is also increasing. Lysogeny, bacteriocinogeny and frequent occurrence of autoplaques are the lytic manifestations of Pseudomonas aeruginosa strains which play a great role in the complexity of solving diagnostic, epidemiological end therapeutical problems connected with infections induced by these microbes. A survey is presented of the importance and utilization of the lytic properties of bacterial strains of Pseudomonas aeruginosa during the differentiation, epidemiological typing and further expansion of therapeutical possibilities in infections caused by Pseudomonas aeruginosa bacteria.

Diagnostic utilization of hemolytically active exosubstances of certain gram-positive bacteria. I. Detection of staphylococcal hemolysins with prepurified preparations of staphylococcal beta-toxin and CAMP-factor of Streptococcus agalactiae.

The authors have modified the one-plate method for the detection of staphylococcal hemolysins. They recommend to use in this method a prepurified form of staphylococcal beta-toxin and of streptococcal CAMP-factor instead of the exclusively beta-tonin-producing strain of Staphylococcus aureus and instead of the intensively CAMP-test positive Streptococcus agalactiae strain, respectively. The authors determined concurrently staphylococcal hemolysins, using a three-plate method in which alpha-antitoxin was employed, to ensure a better evidence of alpha-toxin. A total of 494 staphylococcal strains were examined by both methods. Of this number, 446 Staphylococcus aureus strains were of diverse host origin and 48 were coagulase-negative staphylococcal strains. On the basis of the various hemolytically active staphylococcal toxins, the authors recommend the suggested modification of the one-plate method for their routine detection.

Serological characterization of phages of coagulase-negative staphylococci.

Selected 22 phages of coagulase-negative staphylococci were tested against group-specific antisera for Staphylococcus aureus phages using a neutralization test. Fourteen of the tested phages were classified into the serological group B and one into the group F. All other phages were resistant to the antisera against S. aureus phages but cross-reacted with the antisera produced against themselves. Therefore they could be joined together into a new serological group of the staphylococcal phages.