Comparative in-vitro activity of levofloxacin against Chlamydia spp.
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Anti-Bacterial Agents↗
Biomedical subjects
V Sambri
Publications and source records attributed to V Sambri.
Lyme borreliosis in the Emilia-Romagna region (Italy): identification of the etiological agent.
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Blotting, Western↗
Detection of serum antibodies to CagA and VacA and of serum neutralizing activity for vacuolating cytotoxin in patients with Helicobacter pylori-induced gastritis.
Thirty patients with dyspepsia, with histological diagnosis of gastritis, and with endoscopic diagnosis of peptic ulcer disease (PUD) (n = 13) or nonulcer dyspepsia (NUD) (n = 17) were admitted to the study. Helicobacter pylori vacuolating cytotoxin-producing strains (Tox+) were isolated from 14 (46.7%) patients, whereas non-cytotoxin-producing (Tox-) H. pylori strains were isolated from the remaining patients. Of 30 patients studied, 20 (66.7%) had serum cytotoxin neutralizing activity in vitro. Fourteen patients with Tox+ H. pylori strains showed serum cytotoxin neutralizing activity and serum immunoglobulin G (IgG) and IgA antibodies reactive with both 87-kDa H. pylori vacuolating cytotoxin (VacA) and 128-kDa cytotoxin-associated gene product (CagA) by immunoblotting using native enriched preparations of VacA and CagA proteins from H. pylori culture supernatants as the antigens. A 94-kDa antigen cross-reacting with the 87-kDa VacA protein could be demonstrated in culture supernatant with immune sera from humans and animals. All patients (n = 10) lacking serum neutralizing activity were also negative for IgG or IgA against VacA antigen, whereas 6 of the 10 patients showed IgG serum antibody responses against CagA antigen. The prevalence of antibodies to VacA and CagA antigens was significantly (P < 0.001) higher in patients with gastritis (20 and 26 patients for VacA and CagA, respectively, of 30 patients) than in H. pylori culture-negative controls (0 of 27 for both VacA and CagA) and in randomly selected blood donors (17 and 21 for VacA and CagA, respectively, of 120 subjects). All patients with PUD had antibodies to CagA, whereas 13 of 17 (76.5%) patients with NUD had anti-CagA antibodies. Serum IgG antibodies to VacA were present in 9 (69.2%) patients with PUD of 13 patients and in 11 (64.7%) patients with NUD of 17 patients. Anti-CagA antibodies seemed to correlate better with PUD than anti-VacA antibodies.
Adult↗
Molecular characterisation of Lyme disease borreliae using RAPD analysis and 16S rDNA sequencing.
Here we report the use of random amplified polymorphic DNAs (RAPDs) and sequence analysis of the genes encoding for the small subunit ribosomal RNA (16S rDNA) for the characterisation of Borrelia burgdorferi sensu lato strains recovered from Ixodes ricinus and from Lyme disease patients. All strains examined were assigned to the species Borrelia garinii. However, both RAPDs and 16S rDNAs revealed a level of genetic variation among the strains which appears higher than expected for a bacterial species. In addition, the data obtained agree with the clonal theory applied to Borrelia burgdorferi s.l. for explaining some traits of its epidemiology. According to this theory, particular strains should spread rapidly, leading to the diffusion of bacteria with a particular chromosomal genotype. Our results reveal high genetic variation even among strains isolated in the same period from a restricted geographic area. Moreover, the data here reported indicate that clonal diffusion of antigenic characteristics could also occur.
Borrelia↗
Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells.
In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.
Animals↗
Value of serology (ELISA) for the diagnosis of Helicobacter pylori infection: evaluation in patients attending endoscopy and in those with fundic atrophic gastritis.
In the present study we assessed the diagnostic accuracy of four commercial IgG enzyme-linked immunosorbent assay (ELISA) kits (Autoplate, H.pylori-EIA-Well, Enzygnost, Helori-test) and evaluated the performance of these tests in patients with fundic atrophic gastritis. Serum antibodies to Helicobacter pylori were measured in 70 out-patients attending endoscopy for dyspepsia and 43 patients with non-autoimmune fundic atrophic gastritis. Using the cut-off values recommended by the manufacturers, and comparing serological findings with gastric biopsy results of dyspeptic out-patients attending endoscopy, the four kits showed a sensitivity and specificity, respectively, of 91% and 96%, for Autoplate, 67% and 100% for H.pylori-EIA-Well, 79% and 100% for Enzygnost, and 81% and 96% for Helori-test. Evaluation in patients with atrophic gastritis revealed a high prevalence of antibodies to Helicobacter pylori (84%) and it demonstrated that patients with and those without gastric colonization by this microorganism had a similar rate of seropositivity (76-84% vs 50-78%). In conclusion, our data demonstrate that: a) this assay is a reliable and valid method to detect gastric colonization by Helicobacter pylori; b) positive serum antibody associated with a negative detection of Helicobacter pylori in the gastric mucosa suggests mucosal atrophy; c) patients with fundic atrophic gastritis should be excluded from studies investigating the value of serology in diagnosing Helicobacter pylori infection.
Adult↗
Functional activities of antibodies directed against surface lipoproteins of Borrelia hermsii.
Enriched preparations for mouse polyclonal immunoglobulin G (IgG) antibodies reactive with surface-exposed epitopes (Ab-SEE) of the 22-kDa and 24-kDa membrane lipoproteins of living Borrelia hermsii (HS 1 strain) cells were obtained by an antibody absorption technique using living spirochetes. In vitro, the antibody preparations both inhibited spirochetal growth and were borreliacidal in the presence of complement. The monovalent Fab antibody fragments, prepared from antibody-enriched preparations, did not inhibit the growth of the bacteria, whereas they killed the bacteria in the presence of complement. The two-dimension gel electrophoresis of B. hermsii cells showed that 3H-labeled fatty acids incorporated into the 22-kDa and 24-kDa lipoproteins were resolved into one and three compact spots, respectively. The spots were recognized by the Ab-SEE preparations reactive with the 22-kDa and 24-kDa proteins, by Western blotting.
Animals↗
Borrelia in pigeons: no serological evidence of Borrelia burgdorferi infection.
In order to clarify the supposed involvement of urban pigeons (Columba livia livia) in the epidemiology of Lyme disease, the presence of antibodies against Borrelia burgdorferi and Borrelia anserina in pigeons' sera, collected in 12 areas of northern and central Italy, was evaluated. This evaluation was carried out using a classic immunofluorescence assay (IFA), a surface immunofluorescence assay (SIFA) and a standard Western Blot (WB) assay. A total of 104 out of 3,186 (3.26%) serum samples were positive for both spirochetes when tested by IFA, with titres ranging from 1/40 to 1/1280. All positive specimens showed the same or a higher reactivity against B. anserina than against B. burgdorferi. Of the IFA positive samples, 30 were tested by WB and SIFA to evaluate further the specificity of the antibody response, i.e. to try to clarify against which spirochete the antibodies were raised. The presence of antibodies against the 23 kDa protein exclusive to B. anserina, and against epitopes which are not surface-exposed and which are common to B. anserina and B. burgdorferi, was assessed by WB and SIFA. No serological evidence that B. burgdorferi can infect pigeons was found.
Animals↗
The use of surface immunofluorescence assay (SIFA) in the microbiological diagnosis of Lyme borreliosis: a case report.
In this report we describe the use of a newly developed immunofluorescence technique performed with living spirochetes to detect serum antibody to B. burgdorferi s.l. in a case of early Lyme borreliosis. The immunofluorescence method used (surface immunofluorescence assay: SIFA) proved useful in the serological evaluation of suspected cases of Lyme disease.
Animals↗
Amino acid sequence heterogeneity of the chromosomal encoded Borrelia burgdorferi sensu lato major antigen P100.
The entire nucleotide sequence of the chromosomal encoded major antigen p100 of the European Borrelia garinii isolate B29 was determined and the deduced amino acid sequence was compared to the homologous antigen p83 of the North American Borrelia burgdorferi sensu stricto strain B31 and the p100 of the European Borrelia afzelii (group VS461) strain PKo. p100 of strain B29 shows 87% amino acid sequence identity to strain B31 and 79.2% to strain PKo, p100 of strain B31 and PKo shows 62.5% identity to each other. In addition, partial nucleotide sequences of the most heterogeneous region of the p100 gene of two other Borrelia garinii isolates (PBi and VS286) have been determined and the deduced amino acid sequences were compared with all p100 of Borrelia garinii published so far. We found an amino acid sequence identity between 88.6 and 100% within the same genospecies. The N-terminal part of the p100 proteins is highly conserved whereas a striking heterogeneous region within the C-terminal part of the proteins was observed.
Amino Acid Sequence↗
Glycoprotein patterns in Borrelia spp.
The presence of glycoproteins in several Borrelia species was investigated by the digoxigenin labelling technique. The outer surface proteins A and B of seven isolates of the Lyme disease spirochete B. burgdorferi showed to be major glycosylated proteins. Few minor polypeptides with variable molecular masses were also present, at variance, in B. burgdorferi strains. Minor glycosylated proteins with varying molecular masses have been detected in the relapsing fever borreliae B. hermsii, B. turicatae and B. parkeri. B. turicatae showed also a major glycosylated protein with a molecular mass of approximately 40 kDa. Animal pathogenic borreliae B. anserina and B. coriaceae presented only minor glycosylated proteins with variable molecular masses.
Antigens, Bacterial↗
Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement.
Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii. Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.
Animals↗
Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae.
Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae produced several lipoproteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of bacteria grown in [3H]palmitate. Five major acylated proteins were demonstrated by sequential alkaline and acid hydrolysis. High-pressure liquid chromatography of isolated proteins confirmed that covalently bound radioactivity was represented by fatty acids.
Acylation↗
Adherence of Borrelia burgdorferi and Borrelia hermsii to mammalian cells in vitro.
This study investigated the ability of Borrelia burgdorferi and Borrelia hermsii to attach the surface of several types of in vitro-cultured mammalian cells. Borreliae showed different adhesion efficiencies depending on cell type and temperature. Temperatures both lower and higher than 33 degrees and 37 degrees C respectively, decreased the adhesion of borreliae which preferentially adhere to human fibroblast-like cells. The adhesion process, mediated by structures exposed onto the surface of the microorganisms, also proved to be sensitive to the treatment of mammalian cells with hyaluronidase and sialidase, confirming that carbohydrate receptors are involved in the adhesion of borreliae to eukaryotic cells.
Animals↗
Complement-mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface-protein OspB of Borrelia burgdorferi.
Murine monoclonal antibodies (mAbs) were obtained against the outer-surface-protein OspA and OspB and against the 41-kDa flagellar antigen of Borrelia burgdorferi. The specificity of mAb was determined by the Western blotting technique and the surface association of the antigens was inferred by immunofluorescence of living bacteria. In an in vitro assay in the presence of complement, two mAbs reactive with the Ospa were able to kill borreliae, whereas several mAbs reactive with the OspA as well as with the 41-kDa flagellar protein were not.
Animals↗
Detection of glycoproteins in Borrelia burgdorferi.
The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with 14C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.
Acetylglucosamine↗
Surface immunofluorescence assay for diagnosis of Lyme disease.
A surface immunofluorescence assay (SIFA) was analyzed and compared with a conventional indirect immunofluorescence assay (IFA) and whole-cell enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies to Borrelia burgdorferi in sera from patients with Lyme disease. Fifty-five patients with syphilis and 33 patients with rheumatoid arthritis were used as disease controls. The sensitivity of the SIFA was low during the acute phase of Lyme disease (sera from seven of nine patients presenting with erythema chronicum migrans were negative during the first 2 months of illness); later, seroconversion was observed in all patients at various times during convalescence. Sera from five patients with complicated Lyme disease were strongly positive. SIFA was found to be highly specific, since sera from all patients with secondary or latent syphilis and patients with rheumatoid arthritis did not react in the test. Strong cross-reactivity occurred when these sera were tested in conventional IFA and ELISA; sera from 38 (69%) patients with syphilis were positive by IFA and sera from 51 (93%) patients were positive by ELISA, whereas 7 (21%) and 12 (36%) of the serum samples from patients with rheumatoid arthritis were positive by IFA and ELISA, respectively. Immunoblot analysis of SIFA-positive sera showed that the 31- and 34-kDa outer surface proteins (proteins A and B, respectively) of B. burgdorferi were the major reactive antigens involved in the test. The results support a role for SIFA in the investigation of complicated Lyme disease as well as in the differentiation of Lyme disease from other diseases associated with B. burgdorferi cross-reactive antibodies.
Animals↗
Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.
The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.
Bacterial Proteins↗