Biology and clinical significance of chlamydiae.
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Animals↗
Biomedical subjects
R Marre
Publications and source records attributed to R Marre.
Aggregation substance promotes colonic mucosal invasion of Enterococcus faecalis in an ex vivo model.
BACKGROUND: Bacterial translocation through the gastrointestinal tract is the crucial step in the pathogenesis of intraabdominal infections. We assessed whether aggregation substance (AS), a bacterial adhesin and virulence factor of Enterococcus faecalis, promotes bacterial translocation and colonic mucosal invasion in an ex vivo experiment. METHODS: Colonic mucosa of male Wistar rats was placed in a modified Ussing system. The mucosal side of the chamber was filled with a suspension of E. faecalis OG1X:pAM721 (AS-positive) or E. faecalis OG1X (AS-negative). The serosal side was filled with sterile Dulbecco's modified Eagle's medium. For assessment of colonic mucosal invasion the mucosal side was incubated for 2.5 h with a suspension of AS-positive or AS-negative enterococci. After being washed, a solution of gentamicin and penicillin G in tissue culture medium was added on both sides in order to kill extracellular bacteria. Subsequently, the mucosa was removed from the system, washed, lysed with Triton X-100, and homogenized. Viable intramural bacteria were quantified by plating serial dilutions of the homogenate on Todd-Hewitt broth agar plates. To quantify bacterial translocation samples which were taken at various time points from the serosal side were plated on Todd-Hewitt broth agar plates and colony forming units (CFU) were determined. RESULTS: Invasion of the AS-positive E. faecalis strain OG1X:pAM721 into the colonic mucosa was significantly higher than invasion rates of the AS-negative strain OG1X (2.88 log(10) CFU/ml vs 1.73 log(10) CFU/ml; P = 0.02). However, none of the tested strains was found to translocate from the mucosal to the serosal side within the incubation time of 4 h. CONCLUSIONS: The aggregation substance promotes invasion of E. faecalis into colonic mucosa.
Animals↗
New approaches for diagnosis of infections by intracellular bacteria.
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Bacteria↗
Characterization of the Legionella pneumophila gene ligA.
Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.
Acanthamoeba↗
Decontamination of a Mycoplasma-infected Chlamydia pneumoniae strain by pulmonary passage in SCID mice.
We describe a procedure to eliminate contaminating Mycoplasma from Chlamydia pneumoniae (C. pneumoniae) cultures by pulmonary passage in severe combined immunodeficiency mice (SCID). Four weeks after experimental infection only C. pneumoniae could be cultured from the lungs of the infected animals while Mycoplasma could not be detected any longer, as shown by PCR, culture and transmission electron microscopy (TEM).
Animals↗
Severe Chlamydia pneumoniae infection in patients with neutropenia: case reports and literature review.
Three cases of life-threatening C. pneumoniae infection in patients with acute leukemia and treatment-induced neutropenia are described. Diagnosis was made on the basis of the detection of C. pneumoniae-DNA, complemented by serology. The role of the widely distributed respiratory tract pathogen C. pneumoniae in febrile neutropenia is poorly understood, and studies are needed to estimate the frequency of severe pulmonary infection caused by this agent in patients with neutropenia.
Adolescent↗
Aggregation substance increases adherence and internalization, but not translocation, of Enterococcus faecalis through different intestinal epithelial cells in vitro.
The aggregation substance of Enterococcus faecalis increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.
Adhesins, Bacterial↗
Aggregation substance promotes adherence, phagocytosis, and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst.
The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express beta(2) integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.
Bacterial Adhesion↗
[Community-acquired respiratory tract infections. Current data on the efficacy of various classes of antibiotics and antibiotic resistance of the main prevalent bacteria species].
BACKGROUND: The American Thoracic Society regards fluoroquinolones together with macrolides and doxycycline as first choice antibiotics in the empirical treatment of community acquired pneumoniae in non-hospitalized patients, while the Deutsche Gesellschaft für Pneumology only recommends macrolides and doxycycline for these patients. MATERIAL AND METHODS: In order to find out if the German recommendations still adequately reflect the local resistance situation, we analyzed antibiotic resistance of clinically relevant isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in a multicenter study with special reference to sparfloxacine and ciprofloxacin according to DIN recommendations. RESULTS: In contrast to other European countries and the USA the prevalence of antibiotic resistance in Germany is low. Betalactam antibiotics were effective against more than 95% of strains of Haemophilus influenzae and Streptococcus pneumoniae. However, 10% of the strains of Streptococcus pneumoniae were resistant to tetracycline and 7% resistant to erythromycin. Resistances against ciprofloxacin and sparfloxacine were not detectable. Of both quinolone antibiotics, sparfloxacine was always more active than ciprofloxacin. CONCLUSIONS: These susceptibility data and the known prevalence of other respiratory tract pathogens such as Chlamydia pneumoniae and Mycoplasma pneumoniae which are sensitive to erythromycin and tetracycline therefore support the recommendations of the Deutsche Gesellschaft für Pneumology. However, newer fluoroquinolones with increased activity against pneumococci may be a helpful alternative for patients with persistent and recurrent exacerbations of respiratory infections and patients with relevant underlying diseases or relevant risk factors.
Adult↗
Prevalence of resistance to clarithromycin and its clinical impact on the efficacy of Helicobacter pylori eradication.
BACKGROUND: Triple therapy with a proton-pump inhibitor (PPI) in combination with metronidazole and clarithromycin is the method of choice for eradication of Helicobacter pylori. Failures have been primarily blamed on the development of resistance to clarithromycin. The present study investigated the prevalence and clinical significance of resistance to clarithromycin and metronidazole in determining therapeutic success of both triple therapy as a primary eradication method and high-dose dual therapy in non-responders. METHODS: On the basis of prior therapy, H. pylori-positive patients were assigned to one of two groups in the present prospective study. Group A (n = 93) included patients who had not undergone any prior eradication treatment, whereas group B (n = 15) consisted of patients who had received clarithromycin but in whom eradication had been unsuccessful. All patients underwent endoscopy with biopsy for bacterial culture and resistance studies. Patients in group A were treated with a 7-day regimen of pantoprazole (40 mg twice daily), metronidazole (500 mg twice daily), and clarithromycin (250 mg twice daily), whereas those in group B received omeprazole (40 mg three times a day) and amoxycillin (1000 mg three times a day ) for 14 days. Success of the eradication treatment was ascertained by means of the 13C urea breath test. RESULTS: In group A resistance to clarithromycin and metronidazole was identified in 3 patients (4.9%) and in 14 patients (22.9%), respectively. Eradication proved successful in 78 of 84 patients (92.6%) followed up. Two of the 3 patients with primary clarithromycin resistance and 1 of the 14 patients with metronidazole resistance did not respond to treatment. In group B isolated or combined resistance to clarithromycin was found in seven patients, whereas another four showed isolated resistance to metronidazole. Eradication proved successful in 10 of 13 controlled patients (76.9%) followed up, and only 2 patients reported severe side effects. CONCLUSION: Determination of antibiotic resistance before initiating therapy is not necessary, since primary resistance to clarithromycin is rare. The Italian triple therapy remains a highly effective primary therapeutic method. Further, routine determination of resistance in non-responders also seems to be superfluous because high-dose dual therapy is an effective and well-tolerated second-line therapy regardless of the patients' resistance status.
2-Pyridinylmethylsulfinylbenzimidazoles↗
Analysis of the humoral immune response to Chlamydia pneumoniae by immunoblotting and immunoprecipitation.
Chlamydia pneumoniae is a widely spread agent of respiratory tract infections in humans. A reliable serodiagnosis of the disease is hampered by the poor knowledge about immunodominant antigens in C. pneumoniae infections. We applied a novel strategy to identify immunogenic proteins of C. pneumoniae TW183 combining metabolic radiolabeling of de novo-synthesized chlamydial antigens with immunoprecipitation. By this technique C. pneumoniae antigens of approximately 160, 97 to 99, 60 to 62, 40, 27, and 15 kDa were detected in the vast majority of sera from patients with a current C. pneumoniae infection. By immunoblotting purified elementary bodies of C. pneumoniae TW183 with the same sera, only the 60- to 62-kDa antigen could be detected consistently. Sequential immunoprecipitation performed at different stages of the chlamydial developmental cycle revealed that the 60- to 62-kDa antigen is strongly upregulated after 24 to 48 h of host cell infection and is presented as a major immunogen in both C. pneumoniae-infected patients and mice. We conclude that, due to its high sensitivity and concurrent preservation of conformational epitopes, metabolic radiolabeling of chlamydial antigens combined with immunoprecipitation may be a useful method to reveal important immunogens in respiratory C. pneumoniae infection which might have been missed by immunoblot analysis.
Adolescent↗
Legionella pneumophila invasion of MRC-5 cells induces tyrosine protein phosphorylation.
After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth of L. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [(35)S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L. pneumophila.
Cell Line↗
Legionella pneumophila infection in intratracheally inoculated T cell-depleted or -nondepleted A/J mice.
The inflammatory response and influence of T cell depletion on the pathogenesis of an experimental Legionella infection were studied. A/J mice were infected with 10(6) CFU of Legionella pneumophila intratracheally. With this dose all infected animals survived the infection and bacteria were cleared from lung, spleen, liver, and kidney within 10 to 11 days, leaving no residual changes in the affected organs. Inflammatory cells were recruited into the lung on the second day of infection, reaching a maximum on the third day and filling out predominantly the interstitial areas. During the first 3 days after inoculation, mainly macrophages, B cells, NK cells, and large mononuclear cells of an unknown phenotype were attracted into the lung interstitium, whereas T lymphocytes infiltrated subsequently. During the early phase of infection, serum concentrations of IFN-gamma, TNF-alpha, IL-1beta, IL-4, and IL-6 but not IL-2 increased dramatically. The cytokine secretion decreased on the third day after infection although bacteria were still present in the lung or even disseminated in different organs. Successful clearance of bacteria from the lung was not observed before recruitment of T cells into the lung. In mice depleted of both CD4+ and CD8+ T cells, control of infection was impaired and lethality of infection increased. Depletion of either subset left residual antibacterial mechanisms, which, however, were not sufficient to clear the Legionella as rapidly as in undepleted mice.
Animals↗
Clearance of Chlamydia trachomatis-induced polyserositis in SCID mice requires both CD4+ and CD8+ cells.
To characterize the role of specific lymphocyte subsets in Chlamydia trachomatis infection, we established a murine model using the mouse pneumonitis agent (MoPn) of C. trachomatis and C.B-17 scid/scid (SCID) mice which lack functional B and T cells. After intraperitoneal inoculation with the bacteria, SCID mice developed polyserositis with pleuritis, pericarditis, and perihepatitis. Within 8 weeks post infection, SCID mice succumbed to the disease, whereas immunocompetent congenic C.B-17+/+ mice resolved the infection. Adoptive transfer of immune spleen cells into MoPn-infected SCID mice resulted in a complete elimination of the agent and prevention of polyserositis as measured by quantitative chlamydial culture, direct immunofluorescence and histopathological analysis. Selective reconstitution of MoPn-infected SCID mice with immune B lymphocytes, CD4+ T cells or CD8+ T cells alone did not influence the chlamydial load in the lung and liver of infected SCID animals, resulting in a polyserositis as observed in untreated MoPn-infected SCID mice. However, co-transfer of both CD4+ T cells and CD8+ T cells led to a significant reduction of chlamydiae in quantitative organ culture coupled with unremarkable histopathology. These data confirm that T cell-mediated immune responses are essential for immune protection in chlamydial infection, although total eradication of the agent could not be achieved. Further experiments are needed to stress the importance of a concerted action of B and T lymphocytes, as indicated by the complete protective efficacy of transferred splenocytes.
Adoptive Transfer↗
Bacterial lipopolysaccharide (LPS)-specific antibodies in commercial human immunoglobulin preparations: superior antibody content of an IgM-enriched product.
The anti-LPS antibody content of commercial intravenous immunoglobulins was examined by quantitative ELISA using LPS preparations from Escherichia coli, Klebsiella and Pseudomonas aeruginosa O serotypes occurring most frequently in gram-negative septicaemia. Three IgG products from different manufacturers and one IgM-enriched product were tested. Mean antibody levels were significantly higher in the IgM fraction of the IgM-enriched product compared with 'pure' IgG products, indicating that natural antibodies against bacterial LPS belong primarily to the IgM class. Immunoblotting studies showed that antibody specificities were directed mainly against O side chain epitopes. Antibodies against rough mutant LPS representing various chemotypes were detected in IgG but not in IgM products. The virtual absence of antibodies against Vibrio cholerae LPS indicated that human anti-LPS antibodies result from continuous environmental exposure to gram-negative pathogens. These data support the further development of IgM-enriched preparations for prophylaxis and treatment of gram-negative nosocomial infections.
Antibodies, Bacterial↗
Antibiotic-induced release of endotoxin: in-vitro comparison of meropenem and other antibiotics.
The influence of meropenem, a new carbapenem antibiotic, on cell morphology and in-vitro lipopolysaccharide (LPS) release from Escherichia coli was compared with that of imipenem, ceftazidime, tobramycin and ciprofloxacin. Free and cell-associated LPS was quantified by means of a capture ELISA method based on the recognition of E. coli LPS by monoclonal antibodies. Microscopically, meropenem was found to induce spheroplast formation similar to that seen with imipenem, while ceftazidime and ciprofloxacin induced filament formation. Free and cell-associated LPS levels were low in the presence of meropenem, imipenem, ciprofloxacin and tobramycin, but high in the presence of ceftazidime. Reduced endotoxin release appears to be a common property of carbapenem antibiotics. Morphological changes in bacteria in the presence of antibiotics do not predict their LPS-liberating effect since ciprofloxacin induced low levels of LPS despite causing filament formation.
Anti-Bacterial Agents↗
Sensitivity to sparfloxacin and other antibiotics, of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis strains isolated from adult patients with community-acquired lower respiratory tract infections: a European multicentre study. SPAR Study Group. Surveillance Programme of Antibiotic Resistance.
A survey of resistance to sparfloxacin was carried out in ten European countries, namely Slovakia, France, Germany, Great Britain, Hungary, the Republic of Ireland, Italy, The Netherlands, Portugal and Spain. Respiratory samples were collected from 4297 patients with lower respiratory tract infections and cultured for the presence of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Altogether 2101 strains were isolated and tested for their susceptibility to sparfloxacin, ciprofloxacin, erythromycin, tetracycline and penicillin G (S. pneumoniae) or amoxycillin (H. influenzae and M. catarrhalis). Each country tested strains using methods commonly used in that country, and with breakpoints selected based on those used in that country. Penicillin resistance in pneumococci was seen in those countries in which it had been reported previously, namely Spain, France and Hungary. Only four strains of pneumococci were resistant to sparfloxacin (MIC > or = 2 mg/L), while ciprofloxacin-resistant strains were isolated more frequently, particularly in the Republic of Ireland and Hungary. Almost all of the strains of H. influenzae tested were resistant to erythromycin, (MIC50 > or = 4 mg/L), but all strains were highly sensitive to sparfloxacin (MIC90 < or = 0.06 mg/L). The number of strains of H. influenzae producing beta-lactamase varied between countries, whereas most strains of M. catarrhalis produced beta-lactamase. In M. catarrhalis, erythromycin and tetracycline resistance was rare, but sensitivity to amoxycillin varied. Sparfloxacin was particularly active against H. influenzae and M. catarrhalis, and was the most active compound tested. Overall, the activity of sparfloxacin was greater than that of ciprofloxacin against all three pathogens, and resistance to it was rare.
Anti-Bacterial Agents↗
[Prevention and therapy of bronchial infections. Recommendations of the German Respiratory League of the German Society of Pneumology].
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Anti-Bacterial Agents↗