Grouping of some clinically relevant gram-positive rods by automated fatty acid analysis. Diagnostic implications.

One hundred and ninety-four strains of aerobically growing Gram-positive rods of the genera Corynebacterium, Actinomyces, Arcanobacterium, Erysipelothrix, Listeria, Oerskovia, Nocardia, Rhodococcus, and of unnamed Center for Disease Control (CDC) groups were checked for cellular fatty acid profiles with the microbial Identification System (Microbial ID, Newark, Del., USA). In order to obtain unified data usable for the clinical laboratory, 24 or 48 h sheep blood agar cultures were used. It was thought that grouping and perhaps identification could be aided by this approach. With the aid of numerical analysis, four groups (two consisting of two subgroups each) were established. The discriminatory ability of the scheme, however, was only 77.2%, indicating that grouping of an unknown isolate could be done with some accuracy, but that speciation would still require biochemical testing.

Purification, and biochemical and structural characterization of a fimbrial haemagglutinin of Renibacterium salmoninarum.

Renibacterium salmoninarum was shown to possess peritrichous fimbriae. Electron microscopy of strains FMV 84-01 and ATCC 33209T revealed short, flexible fimbriae less than 2 nm in diameter. These surface appendages were isolated from the bacteria by a procedure involving water extraction and urea solubilization. The fimbrin was purified to homogeneity by Fast Pressure Liquid Chromatography, and shown by SDS-PAGE to be a protein of 57 kDa. Isoelectric focusing under non-denaturing conditions indicated a pI of 4.8. The protein had an amino acid composition rich in glycine, Asx (aspartic acid and asparagine), valine and alanine; methionine was absent. Approximately 33% of the amino acid residues were hydrophobic. Immunoblotting using a polyclonal antiserum raised against whole cells showed that the 57 kDa protein was the immunodominant antigen on the cell surface. Immunogold labelling using polyclonal antibodies raised against the fimbrin revealed an alignment of gold particles along the fimbriae. Purified fimbriae caused agglutination of rabbit erythrocytes and antifimbrial serum inhibited this haemagglutination. Altogether the results indicate that the fimbriae on the surface of R. salmoninarum are responsible for the haemagglutinating activity.

Fatty acid and lipopolysaccharide analyses of three Heliobacterium spp.

The cellular fatty acids from Heliobacterium chlorum, Heliobacterium gestii, and Heliobacillus mobilis were analyzed. The fatty acid contents of the three organisms were essentially the same, consisting of large amounts of branched chain and some mono-unsaturated fatty acids. Neither a phenol-water nor a phenol-petroleum ether-chloroform extraction of whole cells yielded lipopolysaccharide.

Characterization of the Renibacterium salmoninarum haemagglutinin.

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.

On the basic structure of poly(glycerophosphate) lipoteichoic acids.

Poly(glycerophosphate) lipoteichoic acids from 24 Gram-positive bacteria of the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Listeria, Staphylococcus, and the streptococcal pyogenic and oral group were analyzed. The 1,3-linked poly(glycerophosphate) structure was proved by analysis of glycerol and glycerophosphates after acid and alkaline hydrolysis. Using the molar ratios of glycolipid to phosphorus (A) and phosphomonoester to phosphorus after periodate oxidation followed by hydrazinolysis (B) or beta-elimination (C), we show that all lipoteichoic acids contain a single unbranched poly(glycerophosphate) chain and that the chain is uniformly phosphodiester-linked to C-6 of the nonreducing hexopyranosyl residue of the glycolipid moiety. On some chains minor phosphate-containing substituents were detected whose structure remains to be clarified. The lipoteichoic acids of enterococci and listeria strains were separated by hydrophobic interaction chromatography into glycolipid- and phosphatidylglycolipid-containing molecular species. The phosphatidylglycolipid moieties were structurally characterized after liberation from lipoteichoic acids with moist acetic acid. After periodate oxidation of lipoteichoic acids beta-elimination released both phosphatidic acid and the poly(glycerophosphate) chain. This indicates together with the sequence analysis of the released phosphatidylglycolipid that the phosphatidyl residue is located at C-6 of the reducing hexosyl residue of the glycolipid moiety and the poly(glycerophosphate) chain at C-6 of the nonreducing one. Together with earlier observations these results complete the evidence for the structural and possibly biosynthetic relationship between lipoteichoic acids and glycerophosphoglycolipids.

Rapid procedure to determine the DNA base composition from small amounts of gram-positive bacteria.

A universal rapid procedure to determine the DNA base composition (mol% guanine + cytosine) of Gram-positive bacteria is described. Cells of Gram-positive bacteria were lysed with achromo-peptidase and the mol% G + C of their DNAs were determined by using high performance liquid chromatography. One ml of a Gram-positive bacterial suspension which matched MacFarland No. 3 standard turbidity was sufficient to determine the mol% G + C within 3 h.

Rapid diagnosis of bacterial meningitis by the detection of a fatty acid marker in CSF with gas chromatography-mass spectrometry and selected ion monitoring.

A chemical marker of bacterial meningitis was sought by comparing derivatives of sterile cerebrospinal fluid (CSF) with cultures of organisms in spinal fluid and artificial media. The technique of gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) was used, optimised for the analysis of fatty acids. Twenty candidate ions were screened, and an ion of mass: charge ratio (m/e) 268 was chosen for detection in clinical specimens. The origin of this marker is unknown, but it is probably the molecular ion of a C16:1 fatty acid. In 135 clinical specimens of CSF examined, the m/e 268 ion was found to be a useful marker for the common organisms that cause bacterial meningitis, giving a sensitivity of 88% and a specificity of 98%. The method was more rapid and more sensitive than conventional microscopy and culture, but CSF containing coagulase-negative staphylococci, Mycobacterium tuberculosis, Cryptococcus neoformans and some other uncommon pathogens gave inconsistent results. Many organisms produced characteristic ion profiles with multiple-ion monitoring, and this method of chemical analysis holds promise for the rapid diagnosis of bacterial infections to genus or species level.

Cytochrome c-551 from the thermophilic bacterium PS3 grown under air-limited conditions.

A small-sized c-type cytochrome, designated cytochrome c-551, was prepared from membrane fraction of the thermophilic bacterium PS3 grown under air-limited conditions by extraction with cholate, precipitation with polyethylene glycol, and successive chromatographies with DEAE-cellulose and Sephacryl S-200 in the presence of a detergent. The purified sample contained approximately 1 mol of heme c per 10,000 g protein; it showed absorption bands at 551, 522 and 416 nm upon reduction, and a Soret peak at 409 nm upon oxidation. This cytochrome showed a single band of 10 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The isoelectric point of this cytochrome c-551 was pH 4.0. Cytochrome c-551 was suggested to play an important role in the respiratory chain with a terminal oxidase cytochrome o, which is produced under air-limited conditions, since cytochrome c-551 could mediate electron transfer between cytochrome bc1(b6f) complex and cytochrome o, showing quinol oxidase activity.

Structure of a novel glucosamine-containing phosphoglycolipid from Deinococcus radiodurans.

The structure of a major novel lipid from Deinococcus radiodurans has been determined to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho)-3'-O-(alpha-N-acetylglucosaminyl) -N- glyceroyl alkylamine. The lipid was shown to contain a phosphatidic acid backbone by digestion with phospholipase A2 and by hydrolysis with hydrofluoric acid. Using a combination of chemical and NMR spectroscopic techniques, the structure of this lipid was elucidated and compared with that of a similar phosphoglycolipid reported earlier (Anderson, R., and Hansen, K. (1985) J. Biol. Chem. 260, 12219-12223) in which galactose was found in place of N-acetylglucosamine. The fatty acid compositions of the two lipids were similar.

Sandwich immunoassay for the detection of lipoteichoic acid.

A sandwich immunoassay has been developed for the detection of lipoteichoic acid (LTA), a major cell wall constituent of gram-positive bacteria, from whole blood and ISOLATOR supernate. Monoclonal antibodies were produced to purified LTA from Streptococcus mutans, BHT and were further characterized for crossreactivity with gram-positive and negative bacteria and for reactivity to substituted and unsubstituted LTA. Eight monoclonal antibodies were identified that reacted exclusively with gram-positive bacteria. Those antibodies able to capture 3H-LTA were chosen to develop a sandwich immunoassay. The assay has a sensitivity of 0.2 ng LTA/mL in PBS, 0.5 ng/mL in whole blood and 2.0 ng/mL in whole blood that has been processed through the ISOLATOR. Further development of this assay may lead to the rapid detection of LTA from body fluids.

Scanning tunnelling microscopy of biomacromolecules.

When imaging biomacromolecules with a STM, coating of specimens with a conductive layer is a convenient preparation method which provides a good rate of success. Utilizing evaporated platinum/carbon as a coating film we have investigated two biomacromolecules of very different appearance. The first of these is the HPI-layer, a natural two-dimensional protein crystal with a period of 18 nm, which is found on the surface of the bacterium Deinococcus radiodurans. The second specimen is type IV collagen which forms long triple-helical strands approx. 1.5 nm in diameter. The resulting STM pictures compare very well with electron microscopical images.

Gram-positive bacteria: an overview and summary of session.

The more pathogenic gram-positive bacteria present a complex array of surface structures to the human or animal host. The cell wall of Staphylococcus aureus has a pattern of surface proteins; the predominant one is protein A. Virulent S. aureus strains may also produce polysaccharide capsules in vivo that impede opsonization and phagocytosis in the absence of anticapsular antibody. Coagulase-negative staphylococci commonly elaborate an exopolysaccharide slime that may promote adherence to plastic surfaces and interfere with host responses. Structure-function relationships for some antiphagocytic M proteins of group A streptococci are now well understood, and recombinant techniques offer the prospect of multivalent vaccines. The best known surface protein of group B streptococci is the c (Ibc) protein, which stimulates protective antibody in animals and may be an important virulence factor. Monoclonal antibodies to types Ib, II, and III group B streptococci have also confirmed the presence of multiple immunodeterminants on these antiphagocytic polysaccharides. A protein on the surface of pneumococci has been shown to induce protective antibody and to enhance pneumococcal virulence in mice, suggesting a potential alternative or adjunct to pneumococcal polysaccharide vaccines. Listeria also possess a variety of cell surface structures important in pathogenesis. Surface components are, therefore, critical determinants of the interaction of gram-positive bacteria with the host.

Diversity of corrinoids in acetogenic bacteria. P-cresolylcobamide from Sporomusa ovata, 5-methoxy-6-methylbenzimidazolylcobamide from Clostridium formicoaceticum and vitamin B12 from Acetobacterium woodii.

The Co beta-cyanocobamides obtained by cyanide extractions from several acetogenic bacteria were structurally characterized by ultraviolet/visible spectra, proton-nuclear-magnetic-resonance spectra and fast-atom-bombardment mass spectra. p-Cresolycobamide was detected as a major corrinoid from Sporomusa ovata. This 'complete' corrinoid was isolated from an organism for the first time. Instead of the common Co alpha bases of the known and biologically active cobamides, p-cresolylcobamide contained a glycosidically bound cresolyl function that was unable to coordinate to the cobalt of the corrin ring. An additional, previously unknown corrinoid from natural sources, Co alpha-[alpha-(5-methoxy-6-methylbenzimidazolyl)]-Co beta-cyanocobamide, was isolated along with vitamin B12 from Clostridium formicoaceticum. Both homoacetogenic eubacteria were grown on methanol and contained high amounts of corrinoids (greater than 950 nmol/g cell dry mass). Less corrinoid was isolated from Acetobacterium woodii and characterized as vitamin B12.

[Potential use of the monosaccharide composition of bacteria for their identification]. / O vozmozhnosti ispol'zovaniia monosakharidnogo sostava bakterii dlia ikh identifikatsii.

The monosaccharide composition of cell hydrolysates can be used as a criterion for the chemical differentiation of gram-positive bacteria. The monosaccharide composition of six bacterial species belonging to the genus Bacillus has been determined using gas chromatography, mass spectrometry and computers. The qualitative composition was similar, glucose, galactose, ribose and glucosamine being the main components in all of the species. Some Bacillus species differed in their minor components. Although the monosaccharide composition appeared to be homogeneous, bacteria can be identified in terms of their carbohydrate profile using computers. To this end, the monosaccharide composition of bacterial cells is represented as a two-dimensional data file including the qualitative composition of components and the quantity of each component.

Porphyrin test as an alternative to benzidine test for detecting cytochromes in catalase-negative gram-positive cocci.

A total of 66 strains of gram-positive cocci, including 21 catalase-negative members of the family Streptococcaceae and strains of Stomatococcus mucilaginosus, were investigated for the ability to produce porphobilinogen and porphyrin from delta-aminolevulinic acid as an alternative to the benzidine test for detecting the presence of cytochromes. Production of porphobilinogen correlated 100% with membership in the family Micrococcaceae.

Media for study of growth kinetics and envelope properties of iron-deprived bacteria.

Ion-exchange chromatography was used to remove iron from complex and chemically defined laboratory media. The kinetics of metal cation removal from the media was investigated by using atomic absorption spectrophotometry, and the results indicated that over 90% of the iron could be eliminated from certain complex media by this treatment. The treated medium was used for growth studies in a gram-positive and a number of gram-negative organisms that were isolated from infections in humans. High-molecular-weight outer membrane proteins that are known to be induced under iron-depleted growth conditions (iron-regulated membrane proteins) were observed when a number of gram-negative pathogens were cultivated in the treated media. Iron uptake by Staphylococcus aureus varied, depending on the iron content of the medium.

Um meio de cultura de baixo poder de oxi-reduçäo para uso no controle bacteriológico dos canais radiculares: II. Estudo comparativo com caldo infuso cérebro-coraçäo camada baixa oxidado / Culture medium of low oxi-reduction potential for use in bacteriological control of root canals: II. Comparative study with brain-heart infusion medium with low layer oxidated

Um meio de cultura de baixo poder de oxi-reduçäo para uso no controle bacteriológico dos canais radiculares (ZCAR) recentemente estudado em comparaçäo com o caldo glicosado camada baixa oxidado**31, foi avaliado em relaçäo ao caldo infuso cérebro-coraçäo camada baixa oxidado. Foram realizados 100 testes duplos, havendo crescimento, no meio ZCAR, de 17 casos a mais, do que no caldo infuso cérebro-coraçäo camada baixa oxidado. Tal resultado representou maior eficácia do meio ZCAR em 17 por cento que, estatisticamente, mostrou ser significante, pelo teste do X2 (teste do quiquadrado)

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum.

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.

Lethal effect of butylated hydroxyanisole as related to bacterial fatty acid composition.

Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA). Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors. Staphylococcus aureus LP and P. fragi were two of the most resistant species examined; C. perfringens and P. fluorescens were the most susceptible. Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA. There was no obvious relationship between individual fatty acids and susceptibility. The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility. The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility.

Comparison of ribosomes from gram-positive and gram-negative bacteria with respect to the presence of protein S1.

Ribosomes from Gram-positive and Gram-negative bacteria have been analysed for the presence of ribosomal protein S1 by three methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoreaction with E. coli anti-S1 and chromatography on poly (U)-Sepharose. We observed that protein S1 is predominantly present in Gram-negative bacteria in comparison with Gram-positive bacteria. Exceptions are noted in both species.