Cateter venoso central e infección asociada / Catheter associated-sepsis, a prospective study

Para determinar la eficacia del cultivo de la punta del catéter venoso central (CVC), se realizó un estudio prospectivo en el Departamento de Patología Clínica, Servicio de Microbiología del Hospital E. Rebagliati-IPSS de Lima entre enero y marzo de 1993. Treinta y cinco puntas de catéteres, de otros tantos pacientes con diagnóstico de sepsis asociada a CVC, fueron cultivadas mediante la técnica cualitativa en tubos con caldo de tioglicato. La sensibilidad y especificidad encontradas fueron del 60 por ciento. El germen más frecuentemente aislado fue el Estafilococos no patógeno, 32 por ciento del total. Por su baja eficacia, el cultivo de la punta de catéter en caldo debería ser reemplazado por nuevas técnicas.

Application of gas-liquid chromatographic analysis of cellular fatty acids for species identification and typing of coagulase-negative staphylococci.

Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P less than 0.001) higher than that between random isolates of the same species. The correlation coefficient was greater than 97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with less than 95 in all 19 (100.0%) patients with multiple nonidentical isolates. The successful use of the computerized GLC analysis in this study demonstrated its appropriate application for species identification and typing of coagulase-negative staphylococci.

Macrophages produce somnogenic and pyrogenic muramyl peptides during digestion of staphylococci.

Muramyl peptides have a variety of biological effects in mammals, including enhancement of the immune response, sleep, and body temperature. Although mammals lack biosynthetic pathways for muramyl peptides, they are found in mammals and are well known as components of bacterial cell walls. This suggests that phagocytic mammalian cells digest bacterial cell walls and produce biologically active muramyl peptides. Staphylococcal cell walls were radioactively labeled during growth of the bacteria. During the digestion of these radiolabeled bacteria, murine bone marrow macrophages produced low-molecular-weight substances that coeluted chromatographically with the radioactive cell wall marker. Further separation of these substances using reversed-phase high-performance liquid chromatography resulted in the isolation of substances with high specific biological activity. Intracerebroventricular injection of rabbits with these substances induced an increase in slow-wave sleep and body temperature and a suppression of rapid-eye-movement sleep. The characteristics of the biological responses and the chromatographic behavior of the active components are consistent with those of muramyl peptides. The ability of macrophages to tailor muramyl peptides from peptidoglycan may provide an amplification step for the immune response. Muramyl peptides released by macrophages may also act as mediators for various facets of the acute phase response elicited by bacterial infections such as fever and sleep.

Effects of extracellular products from Staphylococcus saprophyticus on human lymphocytes.

Extracellular products from Staphylococcus saprophyticus were isolated and tested for their effect on the lymphoproliferative activity of con A and LPS and on the growth of human lymphocytes. The stimulatory effects of con A or LPS were not affected but a T-cell mitogenic activity was noted after 5 days of incubation. The extracellular material was separated by gel chromatography and the mitogenic activity was shown to be located to the protein fraction.

Typing of coagulase-negative staphylococci isolated from foreign body infections.

Twenty-six coagulase-negative staphylococci isolated from patients with various foreign body infections were characterised using different typing systems. Staphylococcus epidermidis was the most predominant species found. Phage typability was below 50% in all strains. The strains showed differences in surface properties--relative hydrophobicity or hydrophilicity--and ability to adhere to polystyrene with subsequent slime production (adherence tube test). Protein and polypeptide profiles as well as plasmid profiles demonstrated the heterogeneity of the strains. Thus, this preliminary study indicates that all coagulase-negative staphylococci of human origin may become involved in foreign body infections.

A model for the three-dimensional structure of peptidoglycan in staphylococci.

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.

Staphylococcal whole-cell polypeptide analysis: evaluation as a taxonomic and typing tool.

Whole-cell-polypeptide profiles obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used in conjunction with the API-Staph technique to identify different strains of Staphylococcus aureus, S. epidermidis, S. saprophyticus and S. capitis. Complete concordance of results from both techniques was achieved with all strains examined. Visual analysis of the polypeptide patterns and comparison by use of the coefficient of Dice showed minor differences in band pattern between strains of the same species but each species produced a pattern distinguishable from that of any other. The results suggest that although SDS-PAGE can be used to identify staphylococcal species, this type of analysis will not readily provide the basis for a typing method.

Luciferin-luciferase assay of adenosine triphosphate from bacteria: a comparison of dimethylsulphoxide (DMSO) and acetone with other solvents.

The extraction of adenosine triphosphate (ATP) from six strains of bacteria, chosen for differences in cell-wall composition and habitat, was performed. The solvents were two in common use, Tris-ethylenediaminetetraacetate (Tris-EDTA) and trichloroacetic acid (TCA), and two promising, though less utilized solvents, dimethylsulphoxide (DMSO) and acetone. ATP was determined by the luciferin-luciferase reaction. Of the solvents used, DMSO and acetone were the most effective considering the different kinds of bacteria tested and, of these two, DMSO was the most convenient to use. Tris-EDTA was not as effective as the other solvents tested and TCA, which was effective with most strains, gave low yields when used with cultures grown in artificial seawater broth. Internal standards were used to determine if there were substances present that could inhibit the reaction of released ATP with the luciferin-luciferase reagent. Extracts of ATP, stored at -20 degrees C, were stable for up to 3 weeks.

Immunological detection of hCG-like substances in aerobic bacteria of both tumour and non-tumour origin.

Production of hormone-like substances by prokaryotes and unicellular eukaryotes, as well as the presence of a human chorionic gonadotropin-like substance in the serum of individuals with malignancies, have been previously established. To determine the presence and distribution of the production of human chorionic gonadotropin (hCG) among aerobic bacteria, fourteen strains of bacteria of both tumour and non-tumour origin were examined using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and indirect immunoperoxidase techniques. Of the six bacterial strains tested, half of the tumour-isolates produced an hCG-like substance while non-tumour associated strains of the same species did not do so. Virulent strains of two mycobacterial species also gave high positive values while avirulent Mycobacterium tuberculosis strains (H37Ra) yielded low or negative values. The production of hCG-like substances by mycobacteria, for which no tumour association is known, indicates that tumour association is neither a prerequisite nor a requirement for production of hCG. It appears that the presence of hCG-like substances is a variable character among bacterial species, suggesting that it may have been conserved throughout evolutionary history.

Expression of human choriogonadotropin-like material in coagulase-negative Staphylococcus species.

We identified 101 coagulase-negative Staphylococcus strains obtained from different laboratories, the American Type Culture Collection, and our collection, isolated from 23 patients with overt cancer and 34 normal individuals through Kloos and Schleifer conventional methods and the Staph-Ident staphylococcal system (Analytab Products, Plainview, N.Y.). In 40 strains, identity was further verified by DNA-DNA hybridization techniques. Identification revealed 39 S. epidermidis, 22 S. hominis, 8 S. haemolyticus, 9 S. capitis, 5 S. warneri, 5 S. cohnii, 8 S. saprophyticus, and 5 S. xylosus strains, all resident species found in humans. All bacteria were tested for the expression of human choriogonadotropin (hCG)-like material by the indirect fluorescein and peroxidase immunocytochemical labeling techniques by using specific antisera to the whole hormone, to its alpha and beta subunits, to the hCG beta COOH-terminal peptide, and to a monoclonal antibody to the hCG beta. The results demonstrated that the isolates from cancer patients were not unique bacteria, as has been postulated by others; the expression of immunoreactive hCG-like material is a strain, not a species, characteristic; not every bacterial strain isolated from a cancer patient is able to express the material; hCG-producing bacteria do not necessarily indicate the presence of active disease; 20% of the strains that we studied revealed a clonal variation of the expression of hCG-like material or its subunits or both as well as a variable expression of a single hCG epitope, an observation similar to that described for malignant cells; and a specific antiserum to the whole hormone with a high affinity and high sensitivity for immunocytochemistry can be a reliable reagent for screening purposes.

Investigation of Micrococcaceae in a department of cardiac surgery. Biochemical characterization and sensitivity patterns of strains isolated from patients, staff, and air.

A total of 965 strains of Micrococcaceae isolated from 200 patients, personnel, and air in a department of cardiac surgery were classified by means of Baird-Parker's scheme. The majority of strains were identified as Staphylococcus epidermidis (S. epidermidis) biotype 1, but S. epidermidis biotype 4 accounted for c. 25% of isolates from patients post-operatively. Pre-operative isolates were generally sensitive to most antibiotics tested while post-operative strains of coagulase-negative Micrococcaceae from patients and isolates from personnel and air were frequently multiply-resistant. Strains of Staphylococcus aureus (S. aureus) were sensitive or resistant only to penicillin. More patients were colonized with coagulase-negative Micrococcaceae after operation than at admission to the hospital (p less than 0.001), while the frequency of S. aureus carriers was the same before and after operation and equal to the frequency found earlier. The frequency of S. aureus carriers among the personnel, however, was lower than reported earlier (10%). Multiply-resistant strains of S. epidermidis seem to have replaced resistant strains of S. aureus as the predominant hospital saprophyte among Micrococcaceae.

Toxicity of staphylococcal alpha toxin for rabbit alveolar macrophages.

Highly purified staphylococcal alpha toxin was toxic in vitro for rabbit alveolar macrophages. Cytotoxicity, manifested by loss of the ability to exclude trypan blue dye and by morphological evidence of cell necrosis and lysis, was observed after exposure for 4 h to 1 microgram of toxin preparation per ml and after exposure for 8 h to 0.1 microgram of toxin per ml. In addition, exposure to toxin under conditions which did not kill more than 10% of the cells (1 microgram/ml for 1.5 to 2 h) significantly reduced the phagocytic activity of the cells and their ability to respond to an activator of hexose monophosphate shunt activity.

[Membrane protein spectra of staphylococci from a comparative physiological aspect]. / Izuchenie membrannykh belkovykh spektrov stafilokokkov v sravnitel'no-fiziologicheskikom aspekte.

Membrane protein spectra, obtained by electrophoresis in the system polyacrylamide gel--sodium dodecysolfate, were studied in 14 staphylococcal strains with different properties. The proteinograms of membranes were shown to be useful as an additional criterion in the identification of staphylococci. Fractions and their combination characteristic of all the strains and their separate groups were revealed in the protein spectra of staphylococcal membranes.

[Vitamin B6 fluorescence in cells]. / Fluorestsentsiia vitamina B6 v kletke

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.

Chemical structure of the peptidoglycan, its modifiability and relation to the biological activity.

The peptidoglycan is a heteropolymer composed of polysaccharide chains which are cross-linked through short peptides. The polysaccharide moiety (glycan) is made up of beta-1,4 glycosidically linked N-acetylglucosamine and N-acylmuramic acid residues. The carboxyl group of muramic acid is usually substituted by a peptide which consists of alternating L- and D-amino acids. These peptide subunits are cross-linked between the diamino acid in position 3 and the C-terminal D-alanine in position 4 of an adjacent peptide subunit either in a direct way or via an interpeptide bridge (Group A). In some coryneform bacteria the cross-linkage extends from the alpha-carboxyl group of D-glutamic acid in position 2 to D-alanine of a neighbouring peptide subunit (Group B). In the latter case a diamino acid is always found in the interpeptide bridge. A chemical modification of the peptidoglycan may occur in some bacteria due to growth in a quite unbalanced medium. The influence of glycine-rich and glycine-deficient growth medium on the chemical structure of the peptidoglycan of S. aureus will be discussed. Inhibiting concentrations of penicillin, glycine or D-amino acids can also modify the peptidoglycan. Further modification can occur through different extraction procedures which are used to obtain a clean peptidoglycan free of non-peptidoglycan cell wall material. Little is known about the molecular basis of the biological activity. The chemical composition is at least important for the antigenic determinants. The lysozyme susceptibility and the size of the preparation may be other crucial points for the biological activity of the peptidoglycan.

The application of QAE-Sephadex for the purification of two staphylococcal enterotoxins. I. Purification of enterotoxin C2.

A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.