Detection and identification of Citrobacter sedlakii in the Czech Republic.

The biochemical characters of eight strains identified as Citrobacter sedlakii were investigated with the aid of six methods (tube tests, API 50 CH, API 20 E, MICROSCAN, BIOLOG, and CRYSTAL). All the strains were well defined on the basis of biochemical properties investigated with the aid of laboratory-prepared tube tests. The results obtained by the identification kits could not be correctly interpreted. Commercial diagnostic kits should include the reference data necessary for the identification of new Citrobacter species.

[Biochemical indicators of Citrobacter sedlakii]. / Biochemické znaky Citrobacter sedlakii.

The authors describe eight strains identified biochemically as a new species of C. sedlakii in clinical material, surface water and the cutting surface of a melon. The majority of strains was isolated in the Czech and Slovak Republic.

Antitumor activity of bacterial endotoxins and their subunits in in vitro test.

The tumoricidal effect of endotoxins and their subunits of Shigella dysenteriae serovar 1 of both growth forms and certain other representatives of the Enterobacteriaceae family was tested against Németh-Kellner mouse lymphoma cells using an in vitro assay based on the use of sodium chromate solution yielding labelled hexavalent 51Cr ions. The most effective in vitro activity was evidenced in both growth forms by S. dysenteriae 1 lipopolysaccharide-protein complex (LPSP) (76-92%), lipid A and lipoid B isolated from LPS (77-82%) and lipid A and lipoid B from LPSP (53-70%). A direct dependence of the level of the Limulus test and pyrogenicity on the tumoricidal activity of a preparation was not demonstrated. The influence of selected cations (Cu, Fe, Ca, Mg, Zn) bound to selected substances on antitumor activity was monitored. The method of probit analysis is recommended as it enables estimation, based on a number of concentrations, of the regression line of probable effectiveness of a given preparation.

Composition of lipid A in the S and R forms of Shigella dysenteriae serovar 1.

A chemical analysis was performed of lipid A, isolated by acid hydrolysis of the lipopolysaccharide from the S and R forms of Shigella dysenteriae serovar 1. Differences in the moiety of both lipids and sugars were compared. The lipid portion consisted of a homologous series of fatty acids ranging from C12:0 to C18:0 (predominant homologues, C12:0, C14:0 and C16:0) and the homologous series of 3-hydroxy acids ranging from C12:0 to C16:0 (predominant homologue, 3-OH-C14:0). The major sugar portion consisted of D-glucosamine. The toxicity of lipid A in mice (LD50) ranged between 300-400 micrograms/mouse, and values from the Limulus amebocyte lysate assay were recorded as titres of 10(-5) to 10(-6) mg/ml.

Bacterial endotoxins as potential antitumor agents. Tumor mass loss in mice treated with bacterial lipopolysaccharides of Shigella dysenteriae serovar 1.

Natural and modified preparations of lipopolysaccharides and lipopolysaccharide-protein complexes isolated from the S- and R-form of Shigella dysenteriae serovar 1 were found to markedly inhibit the initial growth of mouse solid tumors derived from Németh-Kellner lymphoma, Gardner 6C3HED lymphoma, an ill-defined syngeneic lymphoma of DBA mice (Skalsky lymphoma) and LP-2 plasmacytoma. The biopreparations were given intraperitoneally, most frequently at a dose range from 50 to 200 micrograms per mouse; significant inhibitory effects on tumor growth were evidenced even in mice bearing tumors weighing 113 to 507 mg.

Biological carrier improves passive protection of mice against Pseudomonas aeruginosa infection.

In a model experiment, the use of specific hyperimmune globulins (SHG) alone failed to protect mice against infection with homologous and heterologous P. aeruginosa serotypes. However, the therapeutic potential of SHG dramatically improved after its conjugation with A1(OH)3, used as a biological carrier. The binding of SHG to this carrier proved to be the most effective and stable combination in the treatment of acute pseudomonad infections in mice.

New trends in the use of Al(OH)3-conjugated endotoxins and their subunits from the S- and R-forms of Shigella dysenteriae serovar 1 for model vaccination purposes.

The lipopolysaccharides, lipopolysaccharide-protein complexes and their lipids A, isolated from Shigella dysenteriae 1, exhibited lethal toxicity (LD50 300-400 micrograms per mouse), pyrogenicity (0.01-1.0 microgram), activity in the Limulus test (10(-3)-10(-12) mg ml-1) and produced a positive local Shwartzman reaction. It was found that these substances bound to Al(OH)3 were capable of successfully protecting mice from challenge with both homologous and heterologous infections (up to 90% of mice survived a challenge with LD100), whereas immunization with substances with no biological carrier exhibited very low or zero protective effects. Furthermore, the positive cross-reactivity of lipids A in the agar immunodiffusion test confirms the wide applicability for vaccination purposes.

Tumor necrosis factor and its induction by bacterial endotoxins.

Using suitable immunomodulators (Corynebacterium parvum vaccine, Zymosan or muramyl dipeptide), lipopolysaccharides (LPS) from various members of the family Enterobacteriaceae (Escherichia, Salmonella, Serratia, Shigella) were tested on rabbits in relation to the production of tumor necrosis factor (TNF). TNF was determined by means of the serum titration of L-929 cell cultures in the presence of Actinomycin D, this with resulting titres of 3.2 x 10(3) to 5.1 x 10(4) IU TNF/ml. Analogous titres were noted after the action of denatured LPS (ie LPS subjected to alkaline hydrolysis or H2O2).

Bacterial endotoxins: comparison of mitogenic, polyclonal, antibody-inducing and toxicity activities.

The mitogenic effects on mouse spleen lymphocytes were determined in a large series of commercially available and laboratory-prepared lipopolysaccharides (LPS) obtained from Escherichia, Salmonella, Serratia and Shigella species; part of these LPS preparations was chemically modified prior to testing. In order to establish whether the degree of mitogenic activity corresponds with other biological effects of these preparations, polyclonal activity, capability to induce specific antibody formation and toxicity were determined for selected LPS's with different mitogenic effects. Some of the detoxication procedures used succeeded in reducing the toxicity of LPS while preserving its high mitogenic activitione of the Fe-detoxified preparations of LPS (from the R-form of Shigella dysenteriae serovar 1) exhibited a medium-degree efficacy in all parameters studied. Generally, there was no correlation between the degree of mitogenic activity and the polyclonal and antibody-inducing activities, but in some instances polyclonal activity did correlate with the antibody-inducing activity.

Some requirements for the secretion of tumour necrosis factor and its cytotoxic activity in vitro.

The bacterial lipopolysaccharide (LPS) in vitro-induced secretion of tumour necrosis factor (TNF) by mouse macrophages is described. The highest amount of TNF was produced by DBA/2 mouse peritoneal macrophages activated by a dose of 1 microgram LPS for 3 h. The yield of TNF was higher in the presence of indomethacin during LPS induction. A markedly positive effect on TNF yield of the priming of macrophages in vivo was manifested by proliferation of peritoneal macrophages and increased secretion of TNF as well. No significant effect of hyperthermia (41 degrees C) on TNF secretion could be found. At 41 degrees C, however, TNF cytotoxic activity against the tumour L-929 mouse cells was considerably increased and accelerated, even in the absence of actinomycin D. The maximum cytotoxic activity with or without actinomycin D was reached 15 h later, and it significantly exceeded the value obtained at 37 degrees C at 20 h in the presence of actinomycin D; in the absence of the latter the maximum cytotoxic activity was reached in more than 48 h at 37 degrees C.

Comparative study of culture methods to detect Yersinia enterocolitica serogroup O3 on swine tongues.

Swine tongues were used as a model for evaluation of culture methods for Y. enterocolitica O3. The following three methods were used: Method A consisting of primary culture on DC, McConkey, CIN and SSD agar with additional long-term cold enrichment in PBS and/or selenite broth. Method B--2 min, treatment of samples with 0.5 per cent KOH solution followed by the same cultivation as above and Method C--"cold shock" i.e. incubation of swabs without any medium at -20 degrees C overnight and then inoculated in selenite broth and/or PBS. Methods B and C were approximately twice as effective as Method A. The percentage of detectability ranged from 70 to 90 per cent. CIN agar yielded 20 of 23 positive results when compared with other solid media. A total of 247 smears from pig tongues yielded 60 positive Y. enterocolitica O3 isolations i.e. 27 per cent on average.

Effect of detoxification processes on the interferon-inducing activity of bacterial endotoxins.

The interferon (IFN)-inducing activity of detoxified lipopolysaccharide (LPS) was tested in rabbits treated with LPS preparation derived from Escherichia coli, Salmonella typhi, Salmonella enteritidis and Shigella dysenteriae serovar 1. Of the detoxification procedures used, alkaline hydrolysis, hydroxylaminolysis, formalization, treatment with sodium deoxycholate and the radiodetoxification (fast or slow) methods had no appreciable effects on the IFN-inducing potential of LPS. In contrast, acetylation or prolonged alkaline hydrolysis of LPS resulted in up to a 9-fold reduction of IFN-induction capacity and effects of Cu++ or Fe++ cations bound to LPS were clearly inhibitory (Fe more than Cu).

A case of fatal enterotoxicosis complicated with acute bronchopneumonia caused by Staphylococcus aureus strains producing enterotoxin A.

This is a case report of a female patient 20 years of age who died of congestive heart failure as the result of acute staphylococcal bronchopneumonia resulting from possible aspiration during apparent staphylococcal enterotoxicosis. The diagnosis was supported by the isolation of the same strain of Staphylococcus aureus from the lungs, tonsils, and intestinal contents. Isolates from all three sources produced enterotoxin A, a common food poisoning toxin.

"Slimy eyes phenomenon" in mice intraperitoneally injected with Pseudomonas aeruginosa cells or cell products.

Mice intraperitoneally treated with various Pseudomonas aeruginosa products or lipopolysaccharides of some selected Enterobacteriaceae representatives were found also to react by an increased slime secretion of the eye conjunctivae. The condition, tentatively designated as "Slimy Eyes Phenomenon", started to develop shortly post-treatment, culminated within 24-48 h when the eyes became fully glued up with slime, and receded 48-72 h later, leaving no sequelae for the eye or the general condition of the animal. No such phenomenon has to date been observed in other laboratory animal species.

Long-term preservation of the enteroinvasive Escherichia coli factor responsible for experimental keratoconjunctivitis.

The factor responsible for experimental keratoconjunctivitis (EKC) can be successfully preserved in enteroinvasive Escherichia coli (EIEC) strains for decades. In this respect an adequate freeze-drying technique is essential, whereas the question of protective media appears to be of less importance. The preservation of EKC-producing factor may be associated with the maintenance of some other related properties.

Pseudomonas aeruginosa. II. Experimental acellular vaccine of an endotoxin and anatoxin type.

The results are given of quality evaluation of endotoxin and exotoxin antigens isolated from P. aeruginosa strains. The isolates were tested by both in vitro and in vivo methods. The results of an active protection test on white mice formed the basis for the construction of an experimental Pseudomonas vaccine that protects the immunized animals against infection even by heterologous strains of P. aeruginosa.

[Testing the protective effectiveness of vaccines against infection with Pseudomonas aeruginosa in mink (Lutreola vison)]. / Overování protekcní úcinnosti vakcíny proti infekci Pseudomonas aeruginosa u norku (Lutreola vison).

Infections by Pseudomonas aeruginosa have caused losses on mink farms in recent years, particularly with a clinical manifestation of haemorrhagic pneumonia. This paper includes the first results of the practical use of the Czechoslovak soluble monovaccine of polyvalent action in the treatment of mink infected by Pseudomonas aeruginosa. The action of the vaccine is based on the protective effect of Original Endotoxin Protein (OEP), antigen common to all species of the genus Pseudomonas. After due testing, the vaccine was given to mink. Doses of 50, 200 and 500 micrograms, and in another series 50 and 200 micrograms of the vaccine were tested in subcutaneous administration at 0.2 ml volume in a 7-day interval. No adverse side-effects and reactions were observed in the animals. A protective action was demonstrated, resulting in a higher number of reared mink in comparison with the control groups. After vaccination, titres ranging from 2 to 160 were determined by the indirect haemagglutination method and from 320 to 164,000 by the RIA method. In 1984 the vaccine was used for practical treatment on a mink farm with the stock exposed to Pseudomonas aeruginosa infection (5551 mink were treated). In the vaccinated group losses amounted to 3.1% whereas in the control group the mortality level was above 17%. Vaccination was demonstrated to have a favourable effect and the vaccine was then preventively used on the same farm in the subsequent year; the treatment of 29,350 mink had the required protective effect.

Protective characteristics of the antigen common to Pseudomonas and Vibrio and significance of bacterial endotoxins in protection against selected bacterial infections.

The protective effect of different vaccines against the killing activity of Pseudomonas aeruginosa and Vibrio parahaemolyticus for mice was studied. Cross experiments confirmed the high protective effect of the Pseudomonas common antigen (original endotoxin protein) contained in Pseudomonas vaccine, which, together with other soluble components, substantial afforded protection of mice against vibrios and thus confirmed the antigenic relationship between Pseudomonas and Vibrio. Satisfactory results were also obtained by vaccination with selected bacterial endotoxin lipopolysaccharides derived from some members of the Enterobacteriaceae family, which in some cases afforded 70-90% and in others only a 20-50% protection against Pseudomonas and Vibrio infection. The protective effect was reduced by up to 2/3 was obtained with vaccines subjected to alkaline hydrolysis. A single dose of Propionibacterium parvum cells not followed by specific vaccination afforded up to 50% protection against challenge with P. aeruginosa or V. parahaemolyticus. Special characteristics of the vaccines studied and their protective effects in relation to some immunological aspects of nonspecific resistance are discussed.

Inactivation by polymyxin B of the endotoxin-mediated interferon production in the rabbit.

Polymyxin B (PB) completely or at least significantly inhibited the capacity of Shigella dysenteriae 1 cells and the lipopolysaccharide (LPS) and lipid A (LA) subunits of several bacterial endotoxins to induce interferon (IFN) in rabbits. Animals injected with LPS inactivated by PB to the point of not inducing detectable IFN levels did not develop hyporesponsiveness to secondary IFN induction by a homologous inducer. It was concluded that PB inhibits the IFN-inducing capacity of endotoxin and its subunits as a consequence of binding to the LA-moiety of LPS. The results confirmed the exclusive role of LA as the only IFN-inducing component of Gram-negative bacterial endotoxin.

Relation of enterotoxin production and lipolytic activity in Staphylococcus aureus strains isolated from acute diarrhoeal diseases to clinical course of illness.

Two sets of cases of acute diarrhoeal disease caused by plasma-coagulase-positive Staphylococcus aureus strains were studied. Testing the isolates for staphylococcal enterotoxin (SE) production and for lipolytic activity on egg-yolk agar in relation to the clinical course of the illness showed that only part of the cases could be attributed to the effect of SE and that lipolytic activity apparently also played some pathogenetic role. In one of the sets (205 cases) SE production was found in only 29% of the strains isolated; the clinical course in the corresponding patients was typical of staphylococcal enterotoxicosis. Pronounced lipolytic activity without SE production was demonstrated in 30% of strains; here the clinical course was much milder and protracted. Where both factors were produced (21% of cases), the clinical pattern of enterotoxicosis predominated but was somewhat modified. Two outbreaks in one of which SE production and in the other only clear-cut lipolytic activity were found also differed mutually in the clinical respect. The first displayed a picture of typical staphylococcal enterotoxicosis, the second comprised mild diarrhoeal cases with an incubation period of 6-9 hours, diffuse abdominal pain and no fever. Accordingly, the above observations showed a certain positive correlation between the presence of some staphylococcal exoproducts (SE, lipolytic-activity factor) and the clinical course of the disease. This was particularly striking in infants up to 2 years of age. Lipolytic activity seemed to be associated with mild diarrhoeal staphylococcal disease, although the co-participation of (an)other, so far undetermined factor(s), could not be precluded.