Nonharsh methods of isolation of antigenic complexes of Klebsiella pneumoniae, and analysis of their protective activity

Protective antigenic complexes capable of protecting mice from septic Klebsiella infection can be obtained from Klebsiella pneumoniae. The preparations obtained by the method of aqueous extraction and by the action of hydroxyl amine and autolysis have been found to possess approximately the same protective activity and to protect mice from infection with Klebsiella pneumoniae strains belonging to both homologous and heterologous serotypes.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages]

The purified preparations of Klebsiella bacteriophages, viz. the monovalent preparation of K. pneumoniae bacteriophage and the polyvalent bacteriophage preparation for the treatment of infections caused by K. ozaenae, K. rhinoscleromatis scleromatis and K. pneumoniae sensu lato, have been obtained. The bacteriophage preparations have proved to be nontoxic and safe for laboratory animals after the intraperitoneal injection of these preparations followed by the pathomorphological study of the internal organs of the animals. The clinical study of the newly developed bacteriophage preparations in the course of the treatment of purulent inflammatory diseases in 109 patients has revealed that the preparations are not reactogenic and exhibit sufficient effectiveness in the therapy of ozena, rhinoscleroma and Klebsiella infections with different localization of the infectious process.     

Isolation of different variants of Pseudomonas aeruginosa anatoxin and their characteristics

The conditions for the detoxification of the crude preparations of P. aeruginosa exotoxin A, obtained by the cultivation of strain PA-7 in Martin's broth, have been studied, and the schemes for obtaining nontoxic, stable, specifically antigenic preparations of toxoid from exotoxins A with different degrees of purification have been developed. Toxoid obtained by formalin treatment on the level of a crude preparation with its subsequent purification and additional detoxification with formalin in the presence of lysin has been shown to possess high immunogenic potency. The preparation has been found to induce immune response and to ensure the protection of experimental animals challenged not only with the lethal dose of exotoxin A, but also with P. aeruginosa toxigenic and protease-producing strains.     

Immunological paralysis against type 8 and immunity against type 3 Diplococcus pneumoniae induced by the soluble specific substance of type 8

Brooke, Marcus S. (Massachusetts Institute of Technology, Cambridge). Immunological paralysis against type VIII and immunity against type III Diplococcus pneumoniae induced by the soluble specific substance of type VIII. J. Bacteriol. 90:1296-1303. 1965.-Mice injected with relatively large quantities of the soluble specific substance of type VIII Diplococcus pneumoniae (SVIII) are, on the basis of challenge experiments, paralyzed against type VIII but immune against type III D. pneumoniae. Their sera contain antibodies which react with sheep cells coated with SIII. Unfortunately, it was not possible to develop an in vitro test with SVIII. When the SVIII was treated with a specific SVIII depolymerase before injection into mice, the sera still contained antibodies against SIII and some protection was afforded these mice against type III but not against type VIII challenge. When the SVIII was treated with a specific SIII depolymerase, antibodies were not detectable and the mice were not protected against type III or type VIII challenge. On the basis of these results it is suggested that either the SVIII strain is a hybrid forming both SVIII and SIII molecules or that part of the SVIII chain is altered to consist of repeating units of cellobiuronic acid rather than alternating units of cellobiuronic acid and the disaccharide galactose-glucose. The quantity of SIII in some preparations of SVIII is calculated to be of the order of 0.01%. It is not clear whether all type VIII strains at all times possess the capacity to elicit antibodies which react specifically against SIII, or if this is the property of only some strains and some preparations of these strains.     

Pseudomonas aeruginosa exotoxin

Different P. aeruginosa strains have been found to differ in exotoxin synthesis. The strain isolated at the Mechnikov Research Institute for Vaccines and Sera (Moscow) and newly isolated cultures obtained from patients with the severe course of the infectious process have been found to possess the highest toxigenic activity and to synthesize exotoxins with the most complete set of pathogenically important antigens. The technological scheme for the production of stable exotoxin which can be used for the development of diagnostic, therapeutic and prophylactic preparations against Pseudomonas infections is proposed.     

Production of Escherichia coli-specific hybridomas by using gnotobiotic mice

Monoclonal antibodies provide a rapid and specific means of direct detection of microorganisms in water and food samples. However, monoclonal antibodies specific for some bacteria are difficult to obtain; a good example of such a bacterium is Escherichia coli. Gnotobiotic BALB/c mice immunized with whole-cell preparations of heat-treated strains of E. coli and subjected to high-frequency antigen injection showed a significant increase in the number of specific hybridomas produced. Fusions obtained by using regular BALB/c mice immunized by using standard immunization protocols produced nonspecific hybridomas. Twenty-one stable hybridomas that did not cross-react with Klebsiella pneumoniae ATCC 13883 or Citrobacter freundii 1604770 were obtained from gnotobiotic mice. The bacterial strains were selected for the specificity tests because of their high cross-reactivity, which has been detected in previous fusion experiments. The method of immunization described here offers the potential of improving the production of highly specific hybridomas for bacteria which have been difficult to obtain.     

Production of Escherichia coli-specific hybridomas by using gnotobiotic mice.

Monoclonal antibodies provide a rapid and specific means of direct detection of microorganisms in water and food samples. However, monoclonal antibodies specific for some bacteria are difficult to obtain; a good example of such a bacterium is Escherichia coli. Gnotobiotic BALB/c mice immunized with whole-cell preparations of heat-treated strains of E. coli and subjected to high-frequency antigen injection showed a significant increase in the number of specific hybridomas produced. Fusions obtained by using regular BALB/c mice immunized by using standard immunization protocols produced nonspecific hybridomas. Twenty-one stable hybridomas that did not cross-react with Klebsiella pneumoniae ATCC 13883 or Citrobacter freundii 1604770 were obtained from gnotobiotic mice. The bacterial strains were selected for the specificity tests because of their high cross-reactivity, which has been detected in previous fusion experiments. The method of immunization described here offers the potential of improving the production of highly specific hybridomas for bacteria which have been difficult to obtain.     

Sensitivity to selected beta-lactam antibiotics of clinical strains of Escherichia coli and Klebsiella pneumoniae

The studies aimed at analysing the resistance to some beta-lactam antibiotics among E. coli and K. pneumoniae clinical isolates and at evaluating. The extended spectrum of beta-lactamases (ESBL) production in the isolates. The analysis included 137 E. coli strains and 52 K. pneumoniae strains, isolated from hospitalized patients and out-patients treated in the first trimester of 1998. The strains were identified using the ATB computer system. Antibiotic sensitivity of the isolates was determined by disc-diffusion tests. ESBL production capacity of E. coli and K. pneumoniae strains was estimated by double-disc and ATB BLSA tests. Most of the analysed E. coli strains were found to exhibit significant sensitivity to compound penicillin preparations containing beta-lactam inhibitor (Augmentin, Tazocin) and to the third generation cefalosporins, in contrast, K. pneumoniae strains much more frequently were resistant to the drugs. Among the obtained isolates, 3 (2.2%) E. coli strains and 21 (40.4%) K. pneumoniae strains produced ESBL but all the isolates proved sensitive to imipenem. In evaluation of ESBL production-detecting tests, the double-disc test was found to be more reliable than ATB BLSA test.     

Experimental immunological effectiveness and safety of pyoimmunogen vaccine against Pseudomonas aeruginosa infection

Pyoimmunogen, a polycomponent vaccine against P. aeruginosa infection, has been obtained in laboratory and semi-industrial conditions. The microbial biomass obtained from the strains belonging to O-serotypes (immunotypes) most frequently occurring in clinical practice has been used for producing protective antigens. The preparations have been found to contain proteins (peptides) and carbohydrates in the ratio 6 : 1 to 8 : 1, as well as traces of 2-keto-3-desoxyoctanate, which is indicative of the low content of endotoxin. The immunogenicity of the preparations has been studied experimentally by the active immunization of mice. In these experiments the animals vaccinated in a single injection were found to be protected from challenge with both homologous and heterologous P. aeruginosa strains. The high level of protection from infection caused by toxigenic strain PA-103 was registered. The preparations have low toxicity: LD50 for mice exceeds 2 mg (in protein content): after the multiple administration (7-10 times) of the preparation to mice and rats the weight of the experimental animals was not significantly different from the weight of the control animals.     

Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

Development of a method of obtaining peroxidase conjugates with anti-immunoglobulin for immunoenzyme analysis

The results of the successive stages of preparing peroxidase-labeled anti-immunoglobulins are presented. The schedules for the immunization of animals have been worked out with a view to prepare antisera to human gamma globulin and, subsequently, to isolate antibodies from them by different methods. The preparations obtained with the use of immunosorbent have proved to possess the highest serological activity. As the result of these investigations, a new method of binding, a modification of the glutaraldehyde method, has been proposed. The conjugates obtained by the proposed method are characterized by high serological activity, stability and good reproducibility.     

建立一种反向斑点杂交法检测肺炎支原体23S rRNAV区A2063G基因突变的方法

目的建立检测肺炎支原体（Mycoplasma pneumoniae,MP）23S rRNA V区2063基因型的反向斑点杂交方法,并与PCR产物直接测序法的结果进行比较分析。方法19例自临床咽拭子标本分离培养的MP,用自行设计的反向斑点杂交法检测23S rRNA V区2063基因型,同时对相应序列测序分析。抽提标准菌株FH和127份经PCR测定MP阳性的临床标本基因组DNA,用MP阴性的基因组DNA抽提物5份作阴性对照,用反向斑点杂交法检测23S rRNA V区2063基因型。结果19例分离培养的MP,经反向斑点杂交法检测15株有23S rRNA V区基因A2063G位点突变;4株为2063A,与测序结果一致（Kappa一致性检验,P〉0.05）。经反向斑点杂交法检测,127份MP阳性的基因组DNA抽提物中有122份标本为A2063G位点突变,标准菌株FH和2份DNA抽提物的2063位点碱基为A,还有3份抽提物标本的2063位点碱基既有A又有G,5份阴性对照均无显色。结论反向斑点杂交方法能快速、准确检测临床MP 23S rRNA V区2063基因型。     

Occurrence of clinical isolates of Klebsiella pneumoniae harboring chromosomally mediated and plasmid-mediated CTX-M-15 β-lactamase in a Tunisian hospital

The spread of multidrug-resistant strains of Klebsiella pneumoniae in hospitals is of concern to clinical microbiologists, health care professionals, and physicians because of the impact infections caused by these bacteria have in causing morbidity and mortality. Clinical isolates of K.?pneumoniae have been found to show resistance to third-generation cephalosporins as a result of acquiring extended-spectrum β-lactamase-producing genes, such as bla(CTX-M). Since little is known about the mechanisms of antibiotic resistance observed in Kasserine hospital, Tunisia, this study was undertaken to investigate the mechanisms by which clinical isolates of K.?pneumoniae resist β-lactam antibiotics. Twelve strains of K.?pneumoniae were collected from patients admitted to Kasserine hospital; these isolates showed multiresistance phenotypes. Molecular genetics investigations using polymerase chain reaction, S1 digestion, and pulsed-field gel electrophoresisshowed that bla(CTX-M-15) in association with ISEcp1 is responsible for the resistance of these strains to third-generation cephalosporins. It has been determined that bla(CTX-M-15) is chromosomally mediated and plasmid mediated, which alarming need for infection control to prevent the outbreak of such a resistance mechanism.     

Quinoprotein d-glucose dehydrogenase in Acinetobacter calcoaceticus LMD 79.41: the membrane-bound enzyme is distinct from the soluble enzyme

Abstract The hifH gene from Klebsiella pneumoniae , which codes for the Fe protein component of nitrogenase, was used as a probe to detect nifH homologues in total cellular DNA from 13 obligate methane oxidizing bacteria. All but one of those strains that had previously been shown capable of fixing dinitrogen contained sequences homologous to nifH . DNAs from three of the six non-diazotrophic strains were also found to possess such homology.     

Antibodies against human muscle enolase recognize a 45-kDa bacterial cell wall outer membrane enolase-like protein

Enolase, is a glycolytic enzyme ubiquitous in higher organisms, where it forms tissue specific dimers of isoforms, also found in the cytoplasm of fermentative bacteria. The aim of this work was to identify enolase-like proteins in the cell wall of some Gram-negative bacteria using antibodies against human beta-enolase, an isoenzyme specific to skeletal and heart muscles. Cell wall outer membrane protein (OMP) preparations were obtained from 9 strains of Enterobacteriaceae and one of Pseudomonas aeruginosa. Specific enzymatic enolase activity was detected in the supernatant fractions of cytosolic and inner membrane material, but not in purified OMP preparations. Rabbit polyclonal antibodies specific against human beta-enolase were prepared and purified using immobilized human beta-enolase in affinity chromatography. In SDS-polyacrylamide gel electrophoresis and immunoblotting assay of purified OMP preparations, rabbit anti-enolase antibody interacted specifically with a few OMPs, of which a 45-kDa band also interacted with human sera of patients presenting Buerger disease and atherosclerosis. The most distinct interaction of human sera was observed with a 45-kDa OMP of Klebsiella pneumoniae. This protein was further isolated from K. pneumoniae cell mass in two ways, namely preparative SDS-polyacrylamide gel electrophoresis and specific affinity chromatography using immobilized affinity-purified rabbit antibody raised against human beta-enolase. The data obtained from tandem mass spectrometry tryptic peptide analysis and sequence comparison of human and bacterial enolases using protein databases, could reveal the similarity in the epitopes between membrane enolase-like protein from Klebsiella and human beta-enolase. The results show that the protein present in all studied strains has a common epitope on human beta-enolase. These data raise the question whether such a bacterial protein might be a marker for detecting and monitoring damage to skeletal and heart muscles.     

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.     

Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.     

Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.     

Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.