Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

Structural transformation of human erythrocyte membranes initiated by group specific antibodies

Some changes have been found in the rates of the desintegration by sodium dodecylsulfate of human erythrocite membranes (EM) after incubation with specific group antibodies (GA) from incompatible blood. GA have no effect on the disintegration rates of EM by sodium desoxycholate and Triton X-100. The data are considered as the evidence of the structural transition in EM induced by GA and resulting in modification of integrative coupling forces primarily in the protein components of the membrane. The stopflow technique has been used to investigate the dependence of the desintegration rates in the quick phase (10 to 100 ms) upon the GA concentrations in the incubation medium and upon the incubation time. The obtained curves are distinctly S-shaped that is peculiar of cooperative processes.     

The effect of sodium desoxycholate on the extraction of myocardial creatine phosphokinase and protein.

Rat heart creatine phosphokinase (EC 2.7, 3.2) was extracted in the presence and absence of varying concentrations of sodium desoxycholate and the enzyme activity and total protein content of the extract was determined. Utilization of 0.05 and 0.10% desoxycholate had no effect on the extraction of creatine phosphokinase, while it caused a 70–75% increase in the protein value. Concentrations of 0.5, 0.75 and 1.0% desoxycholate almost doubled the release of creatine phosphokinase. Concomitantly, protein values rose five-to sevenfold. It is suggested that the addition of sodium desoxycholate to a concentration of 0.5% will greatly enhance myocardial creatine phosphokinase release provided the protein content of the extract is not desired as a reference point.     

Indices of phagocytosis of S and R forms of Brucella in patients with acute and chronic brucellosis caused by Brucella abortus

The characteristics of phagocytosis (phagocytic activity and phagocytic index) in patients with acute and chronic brucellosis have been studied with B. abortus S- and R-forms used as its objects. The study has revealed that higher characteristics of phagocytosis with respect to B. abortus S-forms are observed in patients with acute brucellosis, whereas in patients with chronic brucellosis taking a benign course phagocytosis is more intensive in respect of R-forms. In patients with frequent exacerbations of chronic brucellosis differences in the characteristics of phagocytosis with respect to B. abortus S- and R-forms are not statistically valid.     

Bidimensional immunoelectrophoretic study of the antigenic composition of the membrane in various mycoplasma strains]

Membrane antigenic composition of Acheloplasma laidlawii PG9, A. granularum BTS-39, and Mycoplasma fermentans PG 18(G) was determined by means of bidimensional immunoelectrophoresis in the presence of sodium desoxycholate 0.5%. Depending upon the mycoplasma species from which membranes were obtained, 7 to 15 antigens were evidenced. Using sodium desoxycholate presents the advantage over non-ionic detergents to dissolve better the mycoplasmic membrane antigenic complexes. A comparative study of five strains belonging to the above-noted species confirms the serological heterogeneity of the Mycoplasmateles order and shows variability at the membrane antigenic composition level of Acheloplasma laidlawii.     

Differences in the properties of antibodies and natural antiglobulin-homoreactants revealed by the method of human IgG chemical modification]

The naturally occurring antiglobulin factors - homoreactants, contained by human IgG preparations, are inactivated as a result of incubation in a solution of sodium rhodanide (3 - 5 M) and sodium desoxycholate (0.005M). Staphylococcal antitoxin contained by the same human IgG preparations is resistant to the action of the reagents. The data obtained indicate the differences in the structure of homoreactants and antibodies.     

Biological characteristics of Streptococcus pneumoniae strains isolated from children with pneumococcal diseases

The biological properties of 97 S. pneumoniae strains isolated from 92 children with purulent meningitides, meningoencephalitides, pneumonia and otitis, hospitalized at the Leningrad Research Institute of Childhood Infections, were studied. The data obtained in this investigation were indicative of the formation of atypical forms of pneumococci (R-forms, unbalanced growth forms and L-forms) in all clinical forms of pneumococcal infection in children. In purulent meningitides and meningoencephalitides serovars 1 and 6, in pneumonia serovars 1, 3 and 6 and in otitis serovars 6 and 19 played the leading role. The determination of sensitivity to antibiotics showed that the forms under study retained high sensitivity to a number of antibiotics. The appearance of strains resistant to benzylopenicillin was registered. (10%). The isolated strains either possessed low virulence or were avirulent in bioassay on white mice.     

Oxidation of the capsular polysaccharide of pneumoccal type IX by periodate

1. The pneumococcal type IX polysaccharide (polysaccharide S IX) has been oxidized by sodium metaperiodate and reduced by sodium borohydride. Of the constituent monosaccharides, N-acetylglucosamine and N-acetylmannosamine remain unaltered, whereas 40% of the glucose and 90% of the glucuronic acid are oxidized. 2. The effect of oxidation and subsequent reduction on the precipitation of polysaccharide S IX in anti-(pneumococcal) sera is described and interpreted in structural terms. 3. Oligosaccharides produced by oxidation, reduction and hydrolysis with dilute acid have been isolated and partially characterized. 4. The results in this paper and the preceding one (Higginbotham et al., 1972) are used to postulate a possible structure for polysaccharide S IX.     

Surface antigen structure of Neisseria meningitidis. I. The isolation of a serotypic antigenic complex of N. meningitidis strain B16B6 by direct detergent treatment and its immunochemical characteristics

The method for obtaining a serotyping antigenic complex from N. meningitidis B16B6 by their direct treatment with the mixture of detergents (0.5% sodium desoxycholate and 0,5% cholic acid in the proportion 1 : 1) in 0.5 M KCl solution is proposed. Such treatment has been found to increase the yield of the preparation in terms of protein more than 4 times in comparison with earlier methods for obtaining serotyping antigens. The immunochemical study of the preparation has demonstrated its serological specificity and high immunological activity, not inferior to that of serotyping antigenic preparations from group B meningococci, obtained by the heretofore known methods.     

Lumenal plasma membrane of the urinary bladder. II. Isolation and structure of membrane components

A technique has been devised for isolation of lumenal plasma membranes from transitional epithelial cells lining the urinary bladder in rabbits and for subsequent separation of particle-bearing plaque regions from particle-free areas of the membranes. The success of the procedures employed and their effects on the isolates were assessed by electron microscopy of conventional plastic sections, negatively stained preparations, and freeze-etch replicas. When bladders are distended with a solution of 0.01 M thioglycolic acid, which reduces sulfhydryl bridges, cytoplasmic filaments are disrupted, and large segments of the lumenal membranes rupture and float free into the lumen. A centrifugation procedure was developed for isolating a fraction enriched with the large fragments. A comparison of membranes isolated in the presence of thioglycolate with those isolated from epithelial cells homogenized in sucrose medium indicates that thioglycolate has little effect on their fine structure except for the removal of filaments which are normally associated with their cytoplasmic surface. The curved plaques of hexagonally arrayed particles and the particle-free interplaque regions, both characteristic of membranes before exposure to thioglycolate, are well preserved. Subsequent treatment of thioglycolate-isolated lumenal membranes with 1% sodium desoxycholate (DOC) severs many of the interplaque regions, releasing individual plaques in which the particles are more clearly visible than before exposure to desoxycholate. Presumably, DOC acts by disrupting the hydrophobic bonds within the membrane; therefore, this type of cohesive force probably is a major factor maintaining the structural integrity of interplaque regions. This conclusion is consistent with the observation that interplaque regions undergo freeze-cleaving like simple bilayers with a plane of hydrophobic bonding.     

Detection of noncapsular antigens in pneumococcus

Complex antigenic preparations obtained from noncapsular pneumococcal strains were used for the immunization of rabbits and guinea pigs. The injection of the preparations in complete Freund's adjuvant for 5 weeks led to the appearance of antibodies in their sera. The antibodies were detected by the double immunodiffusion test. The preparations obtained from different strains by extraction (with Triton X-100 or sodium deoxycholate) or by disintegration contain common pneumococcal antigens.     

Studies on the Nucleocapsid Structure of a Group A Arbovirus

When Sindbis virus (273S) was treated with sodium desoxycholate, a nonhemagglutinating 136S particle was liberated from the virion, representing the viral nucleocapsid (core). Electron microscopically it appeared as a spherical particle 35 nm in diameter, showing ringlike morphological units 12 to 14 nm in diameter on its surface. When the one- and two-sided images of core particles were correlated, their structure could be demonstrated to have the T = 3 arrangement of 32 hexamer-pentamer morphological units within a symmetrical surface lattice. The core contained a further spherical structure (12 to 16 nm in diameter) which was designated as the central core component. Two proteins were found associated with the core, a third viral protein belonged to the hemagglutinating surface structures. The significance of these findings for virus classification is discussed.     

Noninvasive methods to determine the critical micelle concentration of some bile acid salts

In this work the critical micelle concentrations (cmc) of four bile salts, sodium cholate, sodium glycocholate, sodium deoxycholate, and sodium glycodeoxycholate, are determined and presented. Three independent noninvasive methodologies (potentiometry, derivative spectrophotometry, and light scattering) were used for cmc determination, at 25 degrees C with ionic strength adjusted to 0.10 M with NaCl. Spectrophotometric and potentiometric studies of some bile salts were also executed at various ionic strength values, thus allowing the influence of the ionic strength on the cmc value of the bile salt to be assessed. A critical comparison of the cmc values obtained with data collected from the literature is presented. Furthermore, this work makes an evaluation of the conceptual bases of different methodologies commonly used for cmc determination, since variations in the results obtained can be related mainly to different intrinsic features of the methods used (such as sensitivity or the need to include tracers or probes) or to the operational cmc definition applied. The undoubted definition of the experimental bile salt concentration that corresponds to cmc (operational cmc) is essential since in the case of these amphiphiles the formation of micelles is not as abrupt as in the case of ordinary association colloids. The biphasic nature of their aggregation leads to a "round-shaped" variation of the experimental parameters under analysis, which makes difficult the evaluation of the cmc values and can be responsible for the different results obtained.     

Synthesis and characterisation of a new amphotericin B-methoxypoly(ethylene glycol) conjugate

The reaction of methoxypoly(ethylene glycol)-4-nitrophenyl carbonate with amphotericin B has been used to prepare a new conjugate of amphotericin B (mPEG-AmB). A preliminary screening of in vitro antifungal activity has suggested that mPEG-AmB possesses a similar effect and a similar spectrum of activity as the conventional amphotericin B formulated with sodium desoxycholate.     

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.     

Virulence of Streptococcus pneumoniae strains belonging to different serovars

The study of the virulence of 352 S. pneumoniae strains isolated from patients with inflammatory lung diseases revealed that their virulence depended, to a certain extent, on the state of the polysaccharide capsule of streptococci, as among 299 typed cultures 38.1% were virulent, while out of 53 nontypable strains of these bacteria only 8 strains (15.1%) proved to be highly pathogenic for white mice and all S. pneumoniae R-forms proved to be avirulent. All 11 S. pneumoniae strains under study belonging to serovars 1 and 2 and 87% of the cultures belonging to serovar 3, isolated from patients with inflammatory lung diseases in Leningrad, were highly virulent. The characteristic feature of S. pneumoniae cultures of other serotypes was their wide spectrum of pathogenicity. S. pneumoniae cultures isolated from the spinal fluid of patients with pneumococcal meningitis also differed in their pathogenicity levels but strains highly pathogenic for mice prevailed.     

Evaluation of a rapid immunochromatographic ODK-0901 test for detection of pneumococcal antigen in middle ear fluids and nasopharyngeal secretions

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.     

Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5'' ends

Summary Several pneumococcal bacteriophages showing a morphology similar to that previously described for Cp-1 (Ronda et al. 1981) have been isolated and purified from throat samples taken from healthy children. Three of these phages (Cp-5, Cp-7 and Cp-9) have been studied in detail and compared to Cp-1. The four phages differed in several respects, e.g. size, structural polypeptides, restriction enzyme cleavage patterns, etc. The DNA of Cp-5, Cp-7 and Cp-9 showed protease-sensitive transfecting activity. This, together with the results obtained by electrophoretic analyses as well as by isotopic labelling of these DNAs with [-³²P] ATP and polynucleotide kinase indicated that all these new phages have a protein covalently linked to the 5 ends of their DNAs as in the case of Cp-1 (García et al. 1983). Restriction enzyme cleavage maps of Cp-1, Cp-5, Cp-7 and Cp-9 have been constructed.     

Toxin-Antitoxin Genes of the Gram-Positive Pathogen Streptococcus pneumoniae: So Few and Yet So Many

Summary: Pneumococcal infections cause up to 2 million deaths annually and raise a large economic burden and thus constitute an important threat to mankind. Because of the increase in the antibiotic resistance of Streptococcus pneumoniae clinical isolates, there is an urgent need to find new antimicrobial approaches to triumph over pneumococcal infections. Toxin-antitoxin (TA) systems (TAS), which are present in most living bacteria but not in eukaryotes, have been proposed as an effective strategy to combat bacterial infections. Type II TAS comprise a stable toxin and a labile antitoxin that form an innocuous TA complex under normal conditions. Under stress conditions, TA synthesis will be triggered, resulting in the degradation of the labile antitoxin and the release of the toxin protein, which would poison the host cells. The three functional chromosomal TAS from S. pneumoniae that have been studied as well as their molecular characteristics are discussed in detail in this review. Furthermore, a meticulous bioinformatics search has been performed for 48 pneumococcal genomes that are found in public databases, and more putative TAS, homologous to well-characterized ones, have been revealed. Strikingly, several unusual putative TAS, in terms of components and genetic organizations previously not envisaged, have been discovered and are further discussed. Previously, we reported a novel finding in which a unique pneumococcal DNA signature, the BOX element, affected the regulation of the pneumococcal yefM-yoeB TAS. This BOX element has also been found in some of the other pneumococcal TAS. In this review, we also discuss possible relationships between some of the pneumococcal TAS with pathogenicity, competence, biofilm formation, persistence, and an interesting phenomenon called bistability.     

Synergism of amphotericin B and 2-deoxyglucose against Histoplasma capsulatum yeasts in vitro.

The glucose analogue, 2-deoxyglucose (2DG), enhances both the fungistatic and the fungicidal action of amphotericin B in Fungizone (Squibb) against Histoplasma capsulatum yeasts in vitro. This synergistic effect is more pronounced when the test substances are incorporated in double-diffusion agar plates than in liquid medium. Minimum inhibitory concentrations for 2DG and amphotericin B in Fungizone have been established. The effects of components of Fungizone other than amphotericin B as clinically administered were also studied. Neither sodium desoxycholate nor phosphate buffer had any effect on the test organisms when used in recommended clinical concentrations. The 5% glucose infusion solution greatly enhanced the growth of the pathogen and markedly decreased the effectiveness of amphotericin B. H. capsulatum yeasts quickly became resistant to stepwise increases of Fungizone but not of 2DG. Susceptibility to amphotericin B and to 2DG increased with time within certain limits of exposure. The A (albino) phenotype of H. capsulatum is considerably more resistant to amphotericin B than the B (brown) phenotype, but there are no differences in susceptibilities to 2DG. The potential clinical applications of these studies are discussed, since experimental animals and man are reported to tolerate large amounts of 2DG. The incorporation of 2DG in the polyene antibiotic preparation would render it more effective at lower doses and would decrease clinical toxicity.