Biomedical subjects

A Cloeckaert

Publications and source records attributed to A Cloeckaert.

79 records · Page 5Linked to original sources

PubMedDate not supplied

Monoclonal antibodies to Brucella rough lipopolysaccharide: characterization and evaluation of their protective effect against B. abortus.

We characterized 4 monoclonal antibodies (mAb) specific for rough lipopolysaccharide (R-LPS) of Brucella. mAb were selected by enzyme-linked immunosorbent assay (ELISA) on whole B. abortus 45/20 rough cells and R-LPS from B. melitensis B115 rough cells. Specificity was confirmed by immunoblot analysis using R-LPS and smooth LPS (S-LPS) preparations. Anti-R-LPS revealed the low molecular mass R-LPS molecules below 20.1 kDa in the R-LPS and S-LPS preparations as well as the typical A and M patterns in high molecular mass S-LPS molecules (between 21.5 and 66 kDa) in the S-LPS preparations. An O-polysaccharide-specific mAb revealed only high molecular mass S-LPS molecules in the S-LPS preparation. In ELISA the anti-R-LPS mAb bound better on rough than on smooth B. abortus 544 whole cells, and this was confirmed by immunoelectron microscopy. Protective activity of anti-R-LPS mAb of different isotypes was tested on mice and compared with an S-LPS-specific mAb. Only the IgG3 mAb reduced significantly the splenic infection but did not reach the level of protection conferred by the S-LPS-specific mAb.

Animals↗

PubMedDate not supplied

Overproduction of the Brucella melitensis heat shock protein DnaK in Escherichia coli and its localization by use of specific monoclonal antibodies in B. melitensis cells and fractions.

The Brucella melitensis dnaK gene was amplified by the polymerase chain reaction using primers chosen according to the published sequence of B. ovis and cloned in multiple copy plasmids enabling expression under the control of the Plac promoter. Monoclonal antibodies (mAb) obtained by immunizing mice with B. melitensis B115 cell wall (CW) fraction or by infecting mice with virulent B. melitensis strain H38 and recognizing a 73-kDa band in immunoblotting of the B. melitensis CW fraction reacted with the cloned dnaK gene product and were thus shown to be specific for the heat shock protein DnaK. The anti-Dnak protein mAbs did not react with Escherichia coli control cells or cell lysates and could therefore be specific to Brucella DnaK protein epitopes. These mAbs were further used to study overproduction of the DnaK protein. B. melitensis DnaK overproduction in E. coli resulted in a defect in cell septation and formation of cell filaments. Immunogold labelling with the mAbs and electron microscopy localized the DnaK protein inside as well as outside the E. coli cells, probably resulting from lysis due to toxicity of the overproduced DnaK protein. These results indicated that overproduction of the B. melitensis DnaK protein in E. coli had similar physiological consequences as that of E. coli overproduced in E. coli. The DnaK protein localization in B. melitensis cells was essentially cytoplasmic, as shown by immunoelectron microscopy. Heat shock treatment of these cells resulted in increased binding of mAbs and labelling in the cytoplasm. However, in subcellular fractions the DnaK protein was predominantly found in the cell envelope fraction of B. melitensis, which could perhaps be due to interaction of the DnaK protein with membrane proteins.

Animals↗

PubMedDate not supplied

Growth phase-dependent variations in the outer membrane protein profile of Brucella melitensis.

Changes in Brucella cell envelope protein profiles were investigated with batch cultures of B. melitensis strain 16M in a 2-litre fermenter. Analysis of expression of outer membrane proteins (OMP) (apparent molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa) and heat-shock protein DnaK (73 kDa) was performed with monoclonal antibodies (mAb) and immunoblotting techniques. Synthesis of the 89-kDa OMP and the heat-shock protein DnaK was invariant during B. melitensis growth. Expression of the 10-, 19- and 36-38-kDa minor OMPs was never detected. Variations in profiles of some OMPs, i.e. 25-27-kDa and 31-34-kDa major proteins and 16.5-kDa minor protein, occurred during growth stages, principally at the end of the exponential growth phase. These variations consisted of shifts in apparent molecular masses for the 25-27-kDa and 31-34-kDa OMPs and of peptidoglycan association for the 16.5-kDa OMP. Therefore, whereas the strong association of major OMPs with peptidoglycan was confirmed, results suggested that the 16.5-kDa minor OMP is also a peptidoglycan-associated protein.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Differentiation of Brucella melitensis, B. ovis and B. suis biovar 2 strains by use of membrane protein- or cytoplasmic protein-specific gene probes.

The possibility of differentiating Brucella species and biovars by Southern blot hybridization of agarose gel-electrophoresed HindIII-digested genomic DNA with membrane protein- or cytoplasmic protein-specific gene probes was investigated on 92 reference and field strains representative of all known species and biovars. Based on the RFLP pattern observed, three gene probes, i.e. br25, 39ugpa and omp16 coding for membrane or cytoplasmic proteins differentiated B. melitensis, B. ovis and B. suis biovar 2 strains from each other and from the other Brucella species and biovars. Thus, the use of these specific gene probes could contribute, in addition to previously identified species- or biovar-specific markers, to the molecular identification and typing of Brucella isolates.

Animals↗

PubMedDate not supplied

Mechanisms of quinolone resistance in Salmonella.

As in other Gram-negative bacteria, mechanisms of resistance to quinolones in Salmonella include target gene mutations, active efflux, and decreased outer membrane permeability. However, the exact contribution of these individual mechanisms to resistance, which may nevertheless interplay to reach high-level resistance, has not yet clearly been defined as in other bacteria such as Escherichia coli. This paper reviews the current state of knowledge of quinolone resistance mechanisms in Salmonella by comparison with that of E. coli and future directions of research with particular attention to the recent development of efflux pump inhibitors as possible means of avoiding the emergence and spread of fluoroquinolone resistance.

4-Quinolones↗

PubMedDate not supplied

Molecular characterization, spread and evolution of multidrug resistance in Salmonella enterica typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has emerged during the last decade as a global health problem because of its involvement in diseases in animals and humans. Multidrug-resistant DT104 strains are mostly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines (ACSSuT resistance type). The genes coding for such resistances are clustered on the chromosome. This paper reviews new developments in the characterization of S. enterica Typhimurium DT104, its chromosomal antibiotic resistance genes and their spread among other S. enterica Typhimurium phage types and other S. enterica serovars, the development of specific detection methods, virulence characteristics, and the evolution of multidrug-resistance with regard to the emergence of quinolone resistance.

Animals↗

PubMedDate not supplied

Molecular tools for the characterisation of antibiotic-resistant bacteria.

This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.

Bacteria↗