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Biomedical subjects

R Marre

Publications and source records attributed to R Marre.

At least 73 records · Page 4Linked to original sources

Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia.

We report the case of a woman who had pneumonia due to Chlamydia psittaci. A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence. The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C. psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA. The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease. In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C. psittaci organisms.

Aged↗

In vitro activities of the lichen secondary metabolites vulpinic acid, (+)-usnic acid, and (-)-usnic acid against aerobic and anaerobic microorganisms.

Secondary metabolites of different species of lichen were tested for their activities against a variety of microbial species. While gram-negative rods and fungi were not inhibited by these compounds, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and some anaerobic species (Bacteroides and Clostridium species) were susceptible at the concentrations tested. Vulpinic acid generally was less active than usnic acid, regardless of its stereochemistry. The susceptibility to usnic acid was not impaired in clinical isolates of S. aureus resistant to methicillin and/or mupirocin.

Anti-Bacterial Agents↗

Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1.

The gene flaA, encoding the flagellum subunit protein of Legionella pneumophila serogroup 1, has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12 by using an antiflagellin specific polyclonal antiserum. DNA sequence analysis of the flaA gene revealed the presence of a 1,428-bp open reading frame encoding a protein of 475 amino acids with an apparent molecular mass of 48 kDa that is expressed independently of an E. coli vector promoter. Peptide sequencing of the N terminus of the isolated flagellum subunit protein confirmed that this open reading frame encodes the flagellin. By comparing the FlaA amino acid sequence with those of flagellins of various other bacteria, high degrees of homology in the N-terminal and C-terminal amino acids could be observed. The flaA-specific mRNA was determined to be 1.6 kb in size, the expected size of a monocistronic mRNA. Temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Sequence analysis and primer extension experiments indicated that the transcription of the gene flaA is directed by a sigma 28-like RpoF-FliA factor. By using fliA and fliA+ E. coli K-12 mutants, it was shown that flaA expression in E. coli required the sigma 28 factor. A flaA-specific DNA probe hybridizes with genomic DNA isolated from L. pneumophila and with most of the genomic DNAs from non-L. pneumophila Legionella strains. Two L. pneumophila strains and isolates of Legionella bozemanii and Legionella feeleii (serogroup 1) carry flaA-specific sequences but were not able to produce flagella.

Amino Acid Sequence↗

The lipopolysaccharide of Legionella pneumophila serogroup 1 (strain Philadelphia 1): chemical structure and biological significance.

The lipopolysaccharide (LPS, endotoxin) of Legionella pneumophila serogroup 1 (Philadelphia 1) exhibits peculiar chemical features which may account for its importance as a bacterial virulence factor. The O-chain of this LPS constitutes a homopolymer of an unusual sugar, 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7, 9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid (legionaminic acid) of which about 10-75 residues are present. Due to the lack of free hydroxyl groups, this homopolymer renders the cell surface highly hydrophobic and, therefore, supports adherence to the membrane of target cells including alveolar macrophages. Investigations of the serological specificity of the serogroup 1 LPS revealed a monoclonal antibody (mAb 3/1) which recognizes an epitope located in the environment of the 8-O-acetyl group of legionaminic acid. According to epidemiological studies, this determinant appears to be associated with L. pneumophila virulence. The outer core oligosaccharide of L. pneumophila LPS exhibits also hydrophobic properties due to the presence of N- and O-acetyl groups as well as 6-deoxy sugars. The inner core expresses both similarities and differences as compared to enterobacterial core oligosaccharides in containing Kdo but lacking heptose and phosphate groups. Lipid A possesses some unique structural features since its backbone consists of a bisphosphorylated beta-GlcpN3N-(1-->6)-GlcpN3N disaccharide with only amide-linked acyl groups having 14-22 carbon atoms. Long chain fatty acids [28:0(27-oxo) and 27:0-dioic] possessing the double length as enterobacterial acyl groups, may be responsible for the low endotoxicity of L. pneumophila lipid A.

Animals↗

The structure of the O-specific chain of Legionella pneumophila serogroup 1 lipopolysaccharide.

The O-polysaccharide chain of Legionella pneumophila Philadelphia strain 1 (serogroup 1) lipopolysaccharide was investigated by means of 1H- and 13C-NMR spectroscopy, and chemical analysis. It was found to consist of an alpha-(2-->4) interlinked homopolymer of a 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-nonulos onic acid possessing most likely the D-glycero-L-galacto configuration, representing the first example of an acidic homopolymer of a higher sugar of this class. The ladder-like banding pattern exhibiting small distances between individual bands in the SDS/PAGE is compatible with a monosaccharide repeating unit.

Carbohydrate Conformation↗

Use of a receiver operating characteristic in the evaluation of two commercial enzyme immunoassays for detection of Helicobacter pylori infection.

Two novel commercial IgG enzyme immunoassay (EIA) systems based on acid-glycine-extracted (Pyloriset IgG EIA, Orion Diagnostica) or fast protein liquid chromatography-purified (Cobas Core Anti-H. pylori EIA, Roche Diagnostic Systems) Helicobacter antigens were evaluated in a prospective study involving 127 patients. All patients underwent upper endoscopy with biopsy, and biopsies were examined for the presence of Helicobacter pylori by a rapid urease test, microscopy and culture. Of the 71 patients found to be infected with Helicobacter pylori, 69 (97.2%) and 65 (91.5%) tested positive with the Cobas Core and Pyloriset test, respectively. A detailed receiver operating characteristic analysis of the two tests showed that the Cobas Core assay was more sensitive and specific at every possible cut-off level; gave a better resolution of individual results, indicating a greater fine-sensitivity; and had no grey zone compared to a large grey zone encompassing 13.4% of the serum samples tested with the Pyloriset EIA. The Cobas Core assay appears to be a valuable tool for epidemiological purposes as well as for pre-endoscopic screening of dyspeptic patients.

Antigens, Bacterial↗

A randomized trial of roxithromycin in patients with acute leukemia and bone marrow transplant recipients receiving fluoroquinolone prophylaxis.

Fluoroquinolone prophylaxis in patients with profound neutropenia may be useful for preventing gram-negative bacterial infection, but it is ineffective against gram-positive bacterial infections in the bloodstream, particularly those caused by streptococci and coagulase-negative staphylococci, which appear to have emerged as significant causes of morbidity, decreased treatment efficacy, and the increased costs of empiric antimicrobial therapy. In a prospective, randomized, open trial, we evaluated the efficacy and safety of oral roxithromycin (150 mg twice daily) as additional antibacterial prophylaxis in 131 adult patients with acute leukemia and bone marrow transplant recipients receiving oral ofloxacin. In comparison with patients given ofloxacin alone, fewer patients receiving ofloxacin plus roxithromycin developed bacteremia caused by viridans group streptococci (incidence, 9 versus 0%; P = 0.03), while the incidence of bacteremia caused by other organisms, the incidence of febrile episodes from any cause, the risk of infection-associated complications (including prolonged or secondary fever, pneumonia, septic shock, need for mechanical ventilation, and/or infection-related death), and antimicrobial usage for therapy were comparable between both groups. Adverse events possibly related to the study drugs were slightly more common among the patients receiving the combination treatment (P = 0.05). Although effective for the prevention of streptococcal bacteremia, the addition of roxithromycin to a fluoroquinolone should not be used routinely as a prophylactic regimen in patients with profound neutropenia, but it might be considered and may be useful for cancer patients with a particularly high risk of streptococcal infection and related complications.

Acute Disease↗

Emergence of fluoroquinolone-resistant Escherichia coli at a cancer center.

Prophylactic treatment with fluoroquinolones of patients with profound neutropenia has been found to be useful for preventing gram-negative bacteremia and has become a standard preventive-therapy strategy in many cancer centers, but the development of bacterial resistance is a cause of concern. During the past few years, we have observed an increasing number of patients with leukemia from whom fluoroquinolone-resistant strains of Escherichia coli were isolated. The increase was significant in this patient population, and among patients with other underlying diseases, the rates of isolation of such strains per number of discharges were significantly lower and did not increase. Most of the leukemia case patients (16 of 19) had been pretreated with an oral quinolone (ofloxacin), with cumulative doses until the first isolation of a resistant E. coli strain ranging from 0 to 97.8 g (median, 14.4 g). Repeated isolation of such strains was seen in 8 of 17 patients during a follow-up period of > or = 4 weeks and in 1 of 6 patients during a follow-up period of > or = 16 weeks. Ten patients developed bacteremia (mortality, 1 of 10). On the basis of the number of patients with leukemia admitted to the hematology-oncology service, the incidence of bacteremia caused by fluoroquinolone-resistant E. coli increased from < 0.5% in 1988-1989 and 0.8% in 1990-1991 to 4.5% in 1992-1993 (P < 0.01). MICs for nine isolates obtained from cultures of blood from different patients ranged between 8 and 16 microgram/ml (ciprofloxacin and PD 131628), 8 and 32 microgram/ml (ofloxacin and BAY Y 3118), and 16 and 32 microgram/ml (sparfloxacin) and indicated resistance to trimethoprim-sulfamethoxazole, ampicillin, doxycycline, and chloramphenicol. Of nine isolates obtained from cultures of blood from different patients and that were subjected to genomic DNA typing by pulsed-field gel electrophoresis of XbaI digests, seven were typeable. Among these, four different genotypes were identified, suggesting both the independent development and the horizontal spread of resistant clones of E. coli.

Anti-Infective Agents↗

Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities.

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.

Animals↗

Adhesion and internalization of E. coli strains expressing various pathogenicity determinants.

The adhesion of Escherichia coli to host epithelial cells is the very first step of urinary tract infections followed by the internalization of some bacteria into these cells. These steps are influenced by several surface antigens or products of the pathogen, e.g. fimbriae or adhesins, K antigen, and hemolysin. The bacterial adherence and the internalization of several mutants of an E. coli O18:K5 strain differing in the expression of K5 antigen, hemolysin, and fimbriae were measured using a permanent line of porcine tubuloepithelial cells (LLC-PK1). Strains with K5 antigen were reduced in adherence and internalization in comparison to the K-negative strains. The expression of hemolysin by these strains lead to an increase of adherence and internalization. The internalization of bacteria is influenced mainly by their adherence to the epithelial cells. Thus, the internalization of attached bacteria is rather a kind of endocytosis than an invasion of the bacteria. To confirm this thesis, we investigated the influence of cytoskeletal inhibitors (cytochalasine B, cytochalasine D, colchicine, and chloroquine) on bacterial adherence and internalization. The cytoskeletal inhibitors lead to a significant inhibition of internalization of the bacteria tested. The receptor-mediated endocytosis of bacteria by tubuloepithelial cells may be of importance in the pathogenesis of recurrent urinary tract infection.

Animals↗

Identification and characterization of surface proteins from Staphylococcus saprophyticus.

Staphylococcus saprophyticus, a well known cause of urinary tract infections, possesses several properties, such as hemagglutination of sheep erythrocytes, adherence to various cell types and production of urease, which may be virulence factors. In this contribution, we summarise the present knowledge about recently discovered surface proteins of Staphylococcus saprophyticus, a 95 kDa surface-associated protein (Ssp) and the hemagglutinin, a 160 kDa surface polypeptide. We describe culture conditions conducive to production of these surface proteins and discuss the molecular and clinical implications of our findings.

Bacterial Adhesion↗

Cleavage of tumor necrosis factor-alpha by Legionella exoprotease.

The role of the major secretory protein of Legionella pneumophila, a zinc protease, in Legionella infection is not known. Since an important step of the host reaction in Legionnaires' disease is the production of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages, we studied the interaction of Legionella protease and U-937 cells with respect to TNF-alpha. The Legionella protease was purified by fractionated precipitation, gel filtration and hydrophobic interaction chromatography. The purified enzyme was added to U-937 cells, a promyelocytic cell line. In the supernatants of PMA-treated U-937 cells we found low concentrations of TNF-alpha after incubation with protease. Therefore we pursued the hypothesis of direct enzymatic degradation of TNF-alpha by Legionella protease. Enzymatic cleavage of TNF-alpha was proven by SDS-PAGE, ELISA and TNF-alpha bioassay with L-929 cells. The degradation of TNF-alpha by the Legionella protease was shown in all three systems. Enzymatic degradation of TNF-alpha might be important for the pathogenesis of Legionnaires' disease.

Chromatography, Gel↗

Differential expression of interleukin-6, intracellular adhesion molecule 1, and major histocompatibility complex class II molecules in renal carcinoma cells stimulated with S fimbriae of uropathogenic Escherichia coli.

The production of interleukin-6, intracellular adhesion molecule 1, and major histocompatibility complex class II molecules by a renal carcinoma cell line (ACHN) in response to S fimbriae of uropathogenic Escherichia coli was studied. S fimbriae adhered to ACHN cells and stimulated the production of interleukin-6 and intercellular adhesion molecule 1 but did not affect major histocompatibility complex class II expression by renal carcinoma cells. Our data demonstrate that S fimbriae of E. coli display immunomodulating properties on kidney-derived epithelial cells.

Carcinoma, Renal Cell↗

Growth in serum-free medium improves isolation of Chlamydia pneumoniae.

Infectivity titers were determined for eight Chlamydia pneumoniae strains simultaneously grown in serum-free and serum-supplemented cell culture media. Use of serum-free medium resulted in a 10- to 50-fold increase in the susceptibility of HL cells to chlamydial infection. Comparative primary isolation of a wild-type strain also produced higher inclusion counts in a serum-free environment. Serum-free cultivation is recommended to increase the efficiency of C. pneumoniae isolation from clinical material and to permit elementary body purification without interference caused by serum components.

Chlamydophila pneumoniae↗

Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila.

Two long-chain fatty acids, 27-oxo-octacosanoic acid (28:0(27-oxo)) and heptacosane-1,27-dioic acid (27:0-dioic) were identified for the first time in phenol-chloroform-petroleum ether extracts of Legionella pneumophila, indicating that they are constituents of lipopolysaccharide. The fatty acids were characterised by combined gas-liquid chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Moreover, minor amounts of 29-oxo-triacontanoic (30:0(29-oxo)) acid and nonacosane-1,29-dioic acid (29:0-dioic) as well as 27-hydroxy-octacosanoic acid (28:0(27-OH)) were present in the phenol-chloroform-petroleum ether extract.

Dicarboxylic Acids↗

Genetics of Escherichia coli uropathogenicity: analysis of the O6:K15:H31 isolate 536.

E. coli strain 536 (O6:K15:H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12 linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.

Chromosome Mapping↗

Adherence to and cytotoxicity of Escherichia coli for eucaryotic cell lines quantified by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).

Adherence of Escherichia coli to human epithelial cells (HEp-2) was studied using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) which is cleaved by enzymes of eucaryotic or procaryotic cells to formazan. This method allows to quantify adherence of Escherichia coli to HEp-2 cells and offers the advantage of assaying a large number of eucaryotic cells without using specific antisera or radioactive material. Furthermore, toxic effects of isolated hemolysin cloned in Escherichia coli onto a renal tubular cell line (LLC-PK1) was investigated by this method, showing reduced cellular viability of tubular cells after an incubation period of 10 to 20 min. MTT is therefore considered to be useful to assay the adherence of Escherichia coli to eucaryotic cells and to quantify toxic effects in eucaryotic cells induced by bacterial virulence factors.

Bacterial Adhesion↗

Subtyping of Legionella pneumophila serogroup 1 strains isolated in Germany using monoclonal antibodies.

Legionella pneumophila serogroup 1 (Lp1) strains isolated from patients and hot water supplies in different locations of Germany were subtyped using seven monoclonal antibodies (mabs) in the indirect immunofluorescence test (IFA) and in part, using a dot blot assay. Four of these mabs were produced in Dresden. Three mabs (mab 33G3, mab 32A12 and mab 144c2) were kindly supplied by J. Joly, Quebec, Canada. Altogether, seven antigenic variants were found among Lp1 strains isolated in Germany. Patient strains belonged to the Philadelphia, Benidorm, Knoxville, France, Olda-Heysham and Bellingham subgroups, whereas environmental isolates reacted like the Bellingham, Oxford, Philadelphia, Knoxville and France strains. The majority of patient strains (15 out of 26, 58%) reacted with our mab 3/1 (corresponding to mab 2 of the standard panel), but only 26 out of 118 environmental strains (22%) isolated from 4 of 15 hot water supplies did so (p less than 0.05). The majority of water-borne Lp1 strains reacted with a mab specific of the Bellingham subgroup. Three water systems under study were associated with human legionellosis. Two of them contained Bellingham-like strains, one Philadelphia-like legionellae.

Antibodies, Monoclonal↗