Fully automated quantitation of hepatitis B virus (HBV) DNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: The measurement of HBV DNA levels has become the most direct and reliable method used for accurate diagnosis and prognosis of acute and chronic HBV infection []. Nucleic acid amplification testing (NAT detection) also reduces the pre-seroconversion window period. The method can be used to aid in the management of HBV infection, by the identification of individuals with high levels of viral replication who might benefit from antiviral therapy, monitoring patients on therapy, and identification of the development of resistance. OBJECTIVES: Evaluation of the novel COBAS AmpliPrep/COBAS TaqMan HBV Test, which combines automated extraction of DNA on the COBAS AmpliPrep Instrument (CAP), coupled with real-time PCR on the COBAS TaqMan Analyzer (CTM), thus greatly reducing hands-on time during sample preparation and amplification/detection. The assay fulfils the current requirements: a highly sensitive HBV DNA detection reagent which is calibrated to the International WHO Standard to reliably quantify HBV genotypes A-G in plasma with a very broad measuring range, thereby minimizing laborious repeat testing. STUDY DESIGN: The test was evaluated for sensitivity, dynamic range, precision, clinical and analytical specificity, genotype inclusivity, interfering substances, and correlation with other tests for the detection of HBV DNA (COBAS Amplicor HBV Monitor Test, COBAS TaqMan HBV Test for Use with the High Pure System and VERSANT HBV DNA 3.0 Assay). RESULTS AND CONCLUSION: A fully automated system for sample preparation, amplification and quantitation of HBV DNA was developed that demonstrates a nearly 7-log dynamic range up to 1.1E+08IU/mL, an assay sensitivity (95% hit-rate) of 4-12IU/mL and an equivalent detection of genotypes A-G plus a prevalent pre-core mutant. The results obtained with the new test show a good correlation to titers obtained with other platforms.

Biotin in vitro translation, nonradioactive detection of cell-free synthesized proteins.

In vitro translation of mRNAs into proteins is frequently used to study the coding capacity of RNAs or cDNAs and the functional effects of mutations. In vitro translation assays have traditionally been monitored by following the incorporation of a radiolabeled amino acid into newly synthesized protein. We have optimized an alternative nonradioactive biotin-labeling method. tRNALys is first aminoacylated with lysine, which is then chemically labeled with biotin. When biotin-lysine-tRNALys is added to translation systems, the biotinylated lysine is incorporated into the growing polypeptide chain. After electrophoresis and transfer to a blotting membrane, the biotin-labeled translation products are detected by a chemiluminescent reaction of luminol/iodophenol with streptavidin-coupled horseradish peroxidase. This nonradioactive method yields results equivalent to those obtained using the radioactive method. Biotin-labeled translation products are also biologically functional: (i) biotinylated precursor proteins are transported and processed correctly by dog pancreas microsomes; (ii) transcription factors synthesized by biotin in vitro translation bind specifically to their DNA recognition sequence; and (iii) biotin-modified luciferase keeps its enzymatic activity. The major advantage of the biotin in vitro translation system is that no radioactivity is required, and the method is easy, economical, reproducible and fast--the whole nonradioactive procedure, from translation to detection, can be completed within six hours.

Expression of capsular polysaccharide determines serum resistance in Escherichia coli K92.

The amount of capsular polysaccharide expression has been shown to be the major determinant of serum resistance in Escherichia coli K1. E. coli K92, like K1, is a polymer of sialic acid molecules. It differs from K1 by containing both alpha (2.8) and alpha (2.9) linkages. Four strains of E. coli K92 were tested for serum resistance. Three strains were serum-resistant (50% normal human serum), one strain was moderately serum-sensitive. The serum-resistant strains expressed significantly more capsular polysaccharide than did the serum-sensitive strain. For each of the serum-resistant strains, six mutants were isolated by selection for resistance against infection with a K92-specific bacteriophage. All of the mutants expressed less capsular polysaccharide than the respective wild-type strains. All mutants were more sensitive to serum killing than the wild-type strains. In all groups, the mutants with lowest expression of capsular polysaccharide were highly serum-sensitive. Changes of outer membrane proteins or lipopolysaccharide patterns that were present in some mutants did not correlate with serum resistance properties of the mutants. Furthermore, it was investigated whether the presence of active serum had an influence on capsule expression. In the serum-sensitive strain, the presence of serum induced a significant and concentration-dependent increase of capsule expression. Serum had no effect on capsule expression by the serum-resistant strains. We conclude from the data that the expression of K92 capsular polysaccharide determines serum resistance in the strains examined.

Ultrastructure and biochemical studies of the flagellar sheath of Helicobacter pylori.

Helicobacter pylori flagellar sheaths were isolated by sucrose density-gradient centrifugation and analysed by electronmicroscopy, SDS-PAGE and gas-liquid chromatography. Electronmicroscopy of thin sections of flagella showed an internal electron-dense filament and a surrounding flagellar sheath with the typical bilayer structure of a membrane. The flagellar filaments could be disintegrated by acid treatment and the resulting isolated flagellar sheaths formed vesicles, sometimes with characteristic structures. Centrifugation of flagellar preparations after acid treatment resulted in the enrichment of flagellar sheaths in the pellet. SDS-PAGE analysis of the pellet showed a reduction of the flagellin band and a number of protein bands of 150, 76, 67, 65, 53, 51, 49, 29.5, 18, 17 and 16 kDa. However, there were no major protein bands characteristic for the sheath. Differences between the protein profiles of Sarkosyl-insoluble membranes and flagellar sheaths appeared in the lower M(r) range of 30-14 kDa. Major fatty acids of isolated flagellar sheaths were C 14:0, C 19:0 cyc, C 18:0, and the LPS-specific fatty acids 3-OH C 16:0 and 3-OH C 18:0. The results demonstrate that the flagellar sheaths of H. pylori are membranes and contain LPS and proteins.

Cloning and genetic characterization of a Helicobacter pylori flagellin gene.

Helicobacter pylori produces polar sheathed flagella, which are believed to be essential for the bacterial colonization of the human gastric mucosa. Here we report on the cloning and genetic characterization of a H. pylori gene encoding the subunit of the flagellar filament, the flagellin. Screening of a genomic library of H. pylori with an oligonucleotide probe derived from the N-terminal amino acid sequence of purified flagellin resulted in a recombinant plasmid clone carrying the flagellin-encoding gene flaA on a 9.3 kb Bg/II fragment. The nucleotide sequence of flaA revealed an open reading frame of 1530 nucleotides, encoding a protein with a predicted molecular mass of 53.2 kDa, which is similar in size with the purified flagellin protein in SDS-polyacrylamide gel electrophoresis. Sequence alignment of H. pylori flagellin (FlaA) with other bacterial flagellins demonstrates a high degree of similarity in the amino-terminal and carboxy-terminal regions, including those of the closely related genus Campylobacter (56% overall identity with Campylobacter coli flaA), but little homology in the central domain. Southern hybridizations of chromosomal DNA with flaA-specific probes did not reveal the presence of additional homologous flagellin genes in H. pylori. Sequence analysis of the flaA flanking regions and mapping of the flaA mRNA start site by a primer extension experiment indicated that transcription of the gene is under the control of a sigma 28-specific promoter sequence in H. pylori. The region upstream of the flaA promoter is subject to local DNA modification, resulting in the masking of two out of three closely linked HindIII restriction sites in the chromosome of strain 898-1. Escherichia coli strains harbouring the recombinant plasmid did not produce full-length flagellin and data obtained with FlaA fusion proteins using an E. coli plasmid expression system suggest that a distinct nucleotide sequence in the gene interferes with productive translation of this protein in E. coli.

Lipopolysaccharide alterations responsible for combined quinolone and beta-lactam resistance in Pseudomonas aeruginosa.

Resistant variants of three clinical Pseudomonas aeruginosa isolates were obtained in the presence of aztreonam. The variants exhibited a four- to eightfold increase in the minimal inhibitory concentrations to beta-lactam antibiotics (except imipenem) to quinolones, such as norfloxacin and fleroxacin, chloramphenicol and tetracycline, but not to gentamicin and polymyxin B. beta-Lactamase production was barely detectable in both wild-type strains and the resistant clones. Only ampicillin, cefoxitin and imipenem increased the production of beta-lactamase, whereas various other beta-lactams did not. Penicillin-binding proteins remained unchanged in the aztreonam-resistant clones. The analysis of the outer membrane proteins did not reveal differences in the outer membrane proteins between the wild-type strains and the aztreonam-resistant clones. Two of the three antibiotic-resistant isogenic clones contained less lipopolysaccharides (LPSs) than their corresponding wild-type strains. Moreover, it could be demonstrated that the ratio of 2-keto-3-deoxy octonate to carbohydrate of the LPS changed in any case between the wild-type strains and the aztreonam-resistant clones. These alterations were accompanied by a decrease in surface hydrophobicity of the resistant clones as compared to the wild-type strains. Therefore, quantitative as well as qualitative alterations in the LPS may provide an explanation for the resistant phenotype observed.

Carbapenem resistance in Enterobacter aerogenes is due to lipopolysaccharide alterations.

The extensive characterization of 2 clinical Enterobacter aerogenes isolates resistant to all beta-lactam antibiotics including imipenem revealed that imipenem resistance could not be attributed to overproduction of the chromosomal beta-lactamase; moreover, it was lost after subcultivation and can be thus considered as unstable. The comparison of sensitive and resistant clones revealed that the beta-lactamase in the resistant clones was less inducible in the resistant clones and moreover, there was an altered 2-keto-3-deoxyoctonate/carbohydrate ratio in the resistant clones as compared to the imipenem-sensitive clones, thus suggesting alterations in the lipopolysaccharide (LPS). Neither enzymatic degradation of both imipenem and meropenem nor alterations of the outer membrane proteins could be observed. These findings make it apparent that this type of resistance is likely due to an impaired uptake of the agents due to LPS alterations.

Imipenem resistance in Acinetobacter baumanii is due to altered penicillin-binding proteins.

The comparison of a clinical Acinetobacter baumanii isolate (strain No. 4852/88) and its selected imipenem-resistant (IMR) clone exhibited a complex reorganization of the penicillin-binding proteins (PBPs) with diminished labelling of all PBPs except the 24-kD PBP which showed an increased binding of 14C-penicillin. This protein could not be saturated by preincubation of membranes with imipenem at 8-fold the MIC of imipenem, thus indicating PBP alterations responsible for imipenem resistance. In A. baumanii 4852/88 seven PBPs with the apparent molecular weights of 94, 84, 65, 61, 48, 40 and 24 kD could be detected. beta-Lactamase production was barely detectable in any case and could not be enhanced in the presence of various beta-lactams as the inducer. The outer membrane proteins were found identical in both the wild-type strain and the Im clone. So far, imipenem-resistant A. baumanii isolates have been isolated twice in our diagnostic laboratory; however, no implications on the future relevance of the above findings can be made.

Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori.

Cellular fatty acids, phospholipid fatty acids, and lipopolysaccharide fatty acids of four strains of Helicobacter pylori were analyzed by gas-liquid chromatography. The presence of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, 19-carbon cyclopropane fatty acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid was confirmed. In phospholipids, myristic acid and 19-carbon cyclopropane fatty acid were the major fatty acids. Hydroxy fatty acids and unsaturated fatty acids were not detected or occurred only in small amounts. The major fatty acids of lipopolysaccharides were stearic acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid. Unsaturated fatty acids and 19-carbon cyclopropane fatty acid were not found. The unusual compositions of H. pylori phospholipid and lipopolysaccharide fatty acids may have important implications for the taxonomy, physicochemical membrane properties, and biological activity of lipopolysaccharides.

Influence of beta-lactam antibiotics on serum resistance of K1-positive blood culture isolates of Escherichia coli.

The K1-positive strains of Escherichia coli are a group with considerable clinical importance, serum resistance being a common virulence factor of these strains. In the present paper, the influences of cephaloridine, imipenem, and ceftazidime on the serum resistance of eight serum-resistant K1-positive E. coli blood culture isolates with smooth-type lipopolysaccharide were studied. All strains were rendered more serum sensitive by treatment with subinhibitory concentrations of antibiotics. The amount of the reduction of serum resistance was dependent on the concentration of the antibiotic. Amounts of K1 produced under the influence of the antibiotics were measured and were found to be reduced for almost all strains tested. To further test the hypothesis that antibiotic-induced reduction of serum resistance is mediated by inhibition of K1 expression, isogenic mutants of one strain were produced by selection for resistance against infection with K1-specific bacteriophages. These mutants were found to be highly serum sensitive. We conclude from this study that beta-lactam antibiotics can render K1-positive serum-resistant strains of E. coli highly serum sensitive and that this effect is mediated by inhibition of K1 expression.

The capsular polysaccharide is a major determinant of serum resistance in K-1-positive blood culture isolates of Escherichia coli.

Serum resistance is a major virulence factor of gram-negative bacteria, and K-1 polysaccharide has been shown to contribute to serum resistance in selected strains. To obtain further information about the role of K-1 in serum resistance and to find out whether loss of the ability to produce K-1 can induce loss of serum resistance, we studied the serum resistance of mutants derived from completely serum-resistant, K-1-positive blood culture isolates of Escherichia coli by selection for resistance to infection with K-1 specific bacteriophages. The amounts of K-1 polysaccharide produced by wild-type strains and mutants were measured, and outer membrane protein and lipopolysaccharide (LPS) patterns were analyzed. In each group of mutants, several highly serum-sensitive strains were found. All mutant strains expressed less K-1 than did the corresponding wild-type strains. Mutants that became highly serum sensitive always had less K-1 than did mutants with less-pronounced changes of serum resistance. A few mutants derived from different wild-type strains showed increased expression of outer membrane proteins with molecular weights of about 46,000 and 67,000. All of the wild-type strains examined had smooth-type LPS, and only two mutants had altered LPS structures; alterations of mutants in outer membrane proteins and LPS could not be correlated with alterations of serum resistance. The results indicate that for K-1-positive blood culture strains of E. coli, K-1 expression is a prerequisite for serum resistance, and loss of ability to synthesize K-1 leads to loss of serum resistance.

Role of the outer membrane for quinolone resistance in enterobacteria.

Quinolone-resistant clones were selected from clinical Escherichia coli, Citrobacter freundii and Serratia marcescens isolates in a frequency ranging from 10(-8) to 10(-6). The outer membrane proteins of quinolone-resistant E. coli clones remained unaltered, as was the case for 10 of 11 C. freundii and 4 of 11 S. marcescens clones ('nal B' type). There was no strong relation between alterations of outer membrane proteins and cross-resistance with chemically unrelated compounds such as tetracycline or chloramphenicol; however, tetracycline resistance was observed in some C. freundii clones with unaltered outer membrane proteins ('mar A'). Most of the quinolone-resistant S. marcescens clones can be considered 'nor B' or 'nor C' mutants due to their cross-resistance with other compounds, their altered outer membrane proteins and changes of lipopolysaccharide. In a few cases, subinhibitory quinolone concentrations caused alterations of outer membrane proteins in S. marcescens during mid log phase without development of resistance.

Ultrastructure and chemical analysis of Campylobacter pylori flagella.

Flagella of Campylobacter pylori were analyzed by electron microscopy and purified, and the molecular weight of the flagellin was determined. Isolation of flagella was performed by mechanical shearing from the cell surface, sucrose density gradient centrifugation, and Sepharose CL-4B gel chromatography. The flagella of C. pylori differ from those of other Campylobacter species and of most other bacteria by the presence of a flagellar sheath. The sheath narrows at the end and is linked to a club-shaped terminal structure. The molecular weight of C. pylori flagellin was 51,000.

Interaction of Escherichia coli and macrophages: alteration by treatment of bacteria with beta-lactam antibiotics.

Antibiotics are known to exert an influence on the host-parasite relationship either by impairment of immunocompetent cells or by alteration of the bacterium, such as changes of surface properties or the production of toxins. The main problem in investigating the effect of antibiotics on the surface properties of bacteria consists in morphological changes of bacteria (round cell or filament formation) after treatment e.g. with beta-lactam antibiotics. These changes of morphology lead to problems in the comparison of such bacterial forms with untreated organisms. Therefore, in this study outer membrane vesicles from bacteria were used as a model to investigate the effect of antibiotics on the surface properties of Escherichia coli with regard to the interaction with mouse peritoneal macrophages tested by chemiluminescence reaction. It could be shown that these membrane vesicles induce a luminol dependent chemiluminescence response. Treatment of E. coli with different beta-lactams lead to an increase of the stimulating properties. The relative effectiveness of certain antibiotics depended on the particular E. coli strain. Analysis of the different adhesions involved in the stimulation of macrophages revealed that only mannose-sensitive adhesins were increased after treatment with beta-lactam antibiotics. No stimulation of the membrane-bound NAD(P)H-oxidase could be found following the reaction with outer membrane vesicles. Even the treatment of bacteria with antibiotics did not evoke such a reaction.

Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the quantitative composition of the outer membrane (OM) of two strains of Escherichia coli, on lipid translocation into the OM, and on the production of capsular K1 polysaccharide were studied. The phospholipid/amino acid ratio was reduced in almost all OM preparations from antibiotic-treated bacteria. In one strain, antibiotic treatment increased the lipopolysaccharide/amino acid ratio. The amount of peptidoglycan fragments bound to the OM was increased by all the antibiotics. In pulse-chase experiments with a radioactive lipid precursor, ciprofloxacin, imipenem, and aztreonam inhibited phospholipid translocation into the OM. Furthermore, imipenem, cephaloridine, and ciprofloxacin induced a pronounced reduction of the production of capsular K1 polysaccharide. Thus, antibiotics seem to induce marked changes of the quantitative composition of the cell envelope of E. coli. Possible connections of these data with findings on the influence of antibiotics on functional parameters of the host-parasite relationship such as OM immunogenicity and serum resistance are discussed.

Influence of beta-lactam antibiotics and ciprofloxacin on composition and immunogenicity of Escherichia coli outer membrane.

The effects of subinhibitory concentrations of different beta-lactam antibiotics and one quinolone on the sedimentation of outer membranes (OMs) of Escherichia coli and on the qualitative properties and immunogenicity of OM components were studied. Membranes were prepared by osmotic lysis of plasmolyzed bacteria. OM and cytoplasmic membrane vesicles were separated by sucrose density ultracentrifugation. Two peaks of OM vesicles with different buoyant densities could be isolated; the quantitative contribution of these to the total OM varied, depending upon the growth phase. In early log phase, the OM consisted mainly of lighter material; in late log and stationary phases, the OM consisted mainly of heavier material. Moxalactam, imipenem, and ciprofloxacin inhibited the formation of heavier material in all growth phases. The immunogenicity of OM vesicles was tested in mice by the hemolytic plaque test. The lighter OM material was markedly less immunogenic than the heavier OM material. The vesicles from antibiotic-treated bacteria and those from early-log-phase cells were less immunogenic than vesicles from untreated late-log-phase and stationary-phase bacteria. These changes were found for the immune response against lipopolysaccharides, as well as against OM proteins. Thus, the immunogenicity of OM components seems to be dependent upon the quantitative composition of lighter and heavier compounds, which is strongly influenced by growth phase and treatment with certain antibiotics.

Three-dimensional structure of fimbriae determines specificity of immune response.

We recently described how a fraction of isolated fimbriae from a multifimbriated strain of Escherichia coli O7:K1:H6 (WF96) could be subdivided by sequential disaggregation in disrupting agents into individual subunits with different molecular weights. In this study, antibodies were raised in rabbits against these isolated fimbrial subunits and against purified intact WF96 fimbriae. These sera were tested by Western blot analysis or by enzyme-linked immunosorbent assays for reactivity against the following antigens: intact WF96 fimbriae, dissociated WF96 fimbriae, dissociated and reaggregated WF96 fimbriae, the WF96 21K fimbrial subunit, reaggregated WF96 21K subunits, the WF96 16K subunits, reaggregated WF96 16K subunits, intact fimbriae from four other E. coli strains, and deaggregated fimbriae from these strains. We found that antibody against intact WF96 fimbriae only reacted strongly with intact WF96 fimbriae, depolymerized and reaggregated WF96 fimbriae, or reaggregated fimbrial subunits; no reactions were evident with intact fimbriae from four other E. coli strains. Conversely, antisera prepared against the WF96 16K subunit and against the WF96 21K subunit did not react with intact WF96 fimbriae or with depolymerized and reaggregated WF96 fimbriae, but did react with homologous isolated subunits. One cross-reaction between fimbrial subunits was apparent: anti-WF96 16K subunit bound to a 21K subunit of deaggregated fimbriae, from another E. coli strain. Taken together, the findings indicate that the three-dimensional structure of the fimbrial preparation used to immunize animals determines the specificity of the immune response.

Isolation and separation of physicochemically distinct fimbrial types expressed on a single culture of Escherichia coli O7:K1:H6.

The fimbrial (pili) profile of a single strain of Escherichia coli O7:K1:H6 (WF96) was evaluated. Fimbriae were isolated by sucrose density gradient ultracentrifugation, purified from flagellae by the use of 0.4% sodium dodecyl sulfate (SDS), and separated into distinct fimbrial types. Analysis of the purified WF96 fimbriae by SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands with molecular weights of 16,000 and 21,000. Treatment of the fimbrial mixture with saturated guanidine hydrochloride resulted in the appearance of a third band with a molecular weight of 19,500. The relative susceptibilities of the WF96 fimbrial types to disrupting chemicals (octyl-glucoside, urea, SDS, and guanidine hydrochloride) were assessed by exposure of the fimbrial mixture to each agent, separation of the depolymerized fimbriae from intact fimbriae by gel filtration on Sepharose CL-4B, and identification of the disaggregated fimbrial types by SDS-polyacrylamide gel electrophoresis of column fractions. The physicochemical heterogeneity of the three fimbrial types coexpressed on WF96 was exploited to develop a method for separation of individual fimbriae.