Biomedical subjects

Axel Cloeckaert

Publications and source records attributed to Axel Cloeckaert.

At least 19 recordsLinked to original sources

PubMed2003

Florfenicol resistance in Salmonella enterica serovar Newport mediated by a plasmid related to R55 from Klebsiella pneumoniae.

Florfenicol resistance has emerged over the past few years in multidrug-resistant Salmonella enterica serovars Typhimurium, Agona and Paratyphi B. The floR gene encoding florfenicol resistance is chromosomally located in these serovars within a genomic island of 43 kb called SGI1 (Salmonella genomic island 1). In the present study, we characterized florfenicol resistance in a strain of S. enterica serovar Newport isolated from a turkey in 1990 and that lacked SGI1. Florfenicol resistance was mediated by a conjugative plasmid related to R55 from Klebsiella pneumoniae, which was characterized initially in the 1970s and harbours a gene 95% identical to floR.

Animals↗

PubMed2003

Mobile genes coding for efflux-mediated antimicrobial resistance in Gram-positive and Gram-negative bacteria.

Efflux mechanisms that account for resistance to a variety of antimicrobial agents are commonly found in a wide range of bacteria. Two major groups of efflux systems are known, specific exporters and transporters conferring multidrug resistance (MDR). The MDR systems are able to remove antimicrobials of different classes from the bacterial cell and occasionally play a role in the intrinsic resistance of some bacteria to certain antimicrobials. Their genes are commonly located on the bacterial chromosome. In contrast, the genes coding for specific efflux systems are often associated with mobile genetic elements which can easily be interchanged between bacteria. Specific efflux systems have mainly been identified with resistances to macrolides, lincosamides and/or streptogramins, tetracyclines, as well as chloramphenicol/florfenicol in Gram-positive and Gram-negative bacteria. In this review, we focus on the molecular biology of antimicrobial resistance mediated by specific efflux systems and highlight the association of the respective resistance genes with mobile genetic elements and their distribution across species and genus borders.

Anti-Bacterial Agents↗

PubMed2003

The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice.

Immunogenicity and protective activity against Brucella ovis of detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli, purified rough lipopolysaccharide from B. ovis (R-LPS) and a mixture of rOmp31 extract and R-LPS (rOmp31 extract + R-LPS) were assessed in BALB/c mice. The experimental vaccines were compared with a hot saline extract (HS extract) from B. ovis mainly composed of outer membrane proteins (OMPs) and R-LPS, and known to be protective in mice against a B. ovis infection. Serum antibodies to Omp31 and R-LPS were detected in the corresponding mice using Western blotting with B. ovis whole-cell lysates and ELISA with purified antigens. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. ovis. A significantly lower number of B. ovis colony-forming units in spleens relative to unimmunized (saline injected) controls were considered as protection. Mice immunized with rOmp31 extract or rOmp31 extract mixed with R-LPS developed antibodies that bound to the B. ovis surface with similar titers. Vaccination with rOmp31 extract plus R-LPS provided the best protection level, which was comparable with that given by HS extract. Similar protection was also obtained with rOmp31 extract alone and, to a lesser degree, with R-LPS. Comparisons between groups showed that an extract from E. coli-pUC19 (devoid of Omp31) provided no protection relative to either HS extract, rOmp31 extract or rOmp31 extract mixed with R-LPS. In conclusion, the recombinant Omp31 associated or not with B. ovis R-LPS, could be an interesting candidate for a subcellular vaccine against B. ovis infection.

Animals↗

PubMed2003

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests.

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.

Animals↗

PubMed2003

Fitness cost of fluoroquinolone resistance in Salmonella enterica serovar Typhimurium.

High-level fluoroquinolone (FQ) resistance is still infrequent in salmonellae, compared with other pathogenic enterobacteria. Data provided in this work support the hypothesis that the mechanisms that confer high-level FQ resistance on salmonellae have a prohibitive fitness cost and may thus limit the emergence of highly resistant clones. In vitro mutants that were highly resistant to ciprofloxacin (MIC = 8 and 16 micro g ml(-1)) showed generation times 1.4- and 2-fold longer than their parent strains and were unable to colonize the gut of chickens. Electron microscopy showed an altered morphology for one of these mutants grown to stationary phase. Mutants selected in vivo and exhibiting intermediate resistance to ciprofloxacin (MIC = 2 micro g ml(-1)) also showed growth defects on solid media but had normal generation times in liquid culture and colonized the gut of chickens. After in vitro or in vivo passage in the absence of antibiotic selective pressure, partial reversals of the fitness cost were observed, which were associated with slight decreases in resistance to quinolones and other unrelated antibiotics, but were not linked to the loss of gyrA mutations.

Animals↗

PubMed2003

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis.

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.

Amino Acid Sequence↗

PubMed2003

Characterization of new members of the group 3 outer membrane protein family of Brucella spp.

Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.

Amino Acid Sequence↗

PubMed2003

Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany.

Salmonella genomic island 1 (SGI1) contains an antibiotic resistance gene cluster and has been previously identified in multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, and Paratyphi B. We identified a variant SGI1 antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France. In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function. Thus, this serovar Albany strain represents the fourth S. enterica serovar in which SGI1 has been identified and the first SGI1 example where gene cassette replacement took place in one of its integron structures. The antibiotic resistance gene cluster of serovar Albany strain 7205.00 constitutes a new SGI1 variant; we propose a name of SGI1-F.

Anti-Bacterial Agents↗

PubMed2002

Brucella evolution and taxonomy.

The genus Brucella contains alpha-Proteobacteria adapted to intracellular life within cells of a variety of mammals. Controversy has arisen concerning Brucella internal taxonomy, and it has been proposed that the DNA-DNA hybridization-based genomospecies concept be applied to the genus. According to this view, only one species, Brucella melitensis, should be recognized, and the classical species should be considered as biovars (B. melitensis biovar melitensis; B. melitensis biovar abortus; etc.). However, a critical reappraisal of the species concept, a review of the population structure of bacteria and the analysis of Brucella genetic diversity by methods other than DNA-DNA hybridization show that there are no scientific grounds to apply the genomospecies concept to this genus. On the other hand, an enlarged biological species concept allows the definition of Brucella species that are consistent with molecular analyses and support the taxonomical standing of most classical species. Both the host range as a long-recognized biological criterion and the presence of species-specific markers in outer membrane protein genes and in other genes show that B. melitensis, B. abortus, B. ovis, B. canis and B. neotomae are not mere pathovars (or nomenspecies) but biologically meaningful species. The status of B. suis is, however, less clear. These approaches should be useful to define species for the marine mammal Brucella isolates, as illustrated by the grouping of the isolates from pinnipeds or from cetaceans by omp2 gene analysis. It is shown that a correct Brucella species definition is important to understand the evolution of the genus.

Biological Evolution↗

PubMed2002

Major outer membrane proteins of Brucella spp.: past, present and future.

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 and 25-27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.

Amino Acid Sequence↗

PubMed2002

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis.

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.

Animals↗

PubMed2002

Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams.

To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams.

Animals↗

PubMed2002

Identification of Brucella melitensis vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene.

The live attenuated strain B. melitensis Rev.1 is considered the best vaccine available for the prophylaxis of brucellosis in sheep and goats. The Rev.1 vaccine was obtained in the 1950s by a two-step selection involving firstly streptomycin resistance and dependence and secondly reversion of dependence but keeping streptomycin resistance. Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. Nucleotide sequencing revealed one mutation in the rpsL gene of vaccine strain Rev.1 compared to that of reference strain 16M leading to an amino acid Pro-to-Leu change at codon position 91 (Pro91Leu). This mutation resulted also in the lack of a NciI restriction site in the gene. PCR-restriction fragment length polymorphism (PCR-RFLP) using NciI applied to a large number of Brucella reference and field strains showed that the mutation detected was specific of vaccine strain Rev.1.

Bacterial Vaccines↗

PubMed2002

Brucella abortus vaccine strain RB51 produces low levels of M-like O-antigen.

Brucella abortus RB51 is a rough (R) stable vaccine strain used in cattle and is believed to be devoid of O-side chain. We analyzed by use of a panel of monoclonal antibodies (MAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes the O-chain expression in strain RB51. Two MAbs specific for the C/Y (A=M) and C (M>A) epitopes showed low bindings in ELISA to strain RB51. O-chain expression was further confirmed by Western blot after SDS-PAGE of strain RB51. In particular, the MAb of C (M>A) specificity, showing preferential binding to M-dominant smooth (S) Brucella strains, revealed in strain RB51 a typical smooth-lipopolysaccharide (S-LPS) pattern which resembled that of M-dominant S-LPS. Thus, the results clearly show that strain RB51 produces low levels of M-like O-antigen.

Animals↗

PubMed2002

Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA.

A study was conducted to evaluate the immunogenicity of the Brucella melitensis ribosome recycling factor (RRF)-homologous protein (CP24). The CP24 gene was cloned, expressed in Escherichia coli and purified. The resulting purified recombinant protein (rCP24) produced delayed-type hypersensitivity (DTH) reactions in B. melitensis-infected mice but not in naive controls. Thus, we decided to characterise the immune responses generated with DNA vaccination (pcDNACP24) or immunisation with the rCP24 in adjuvant. Animals injected with pcDNACP24 exhibited a dominance of IgG2a to IgG1 while mice injected with rCP24 developed a higher response of IgG1 than IgG2a. Both immunisation protocols were capable of eliciting CP24-specific gamma interferon (IFN-gamma) producing cells. Spleen cells from pcDNACP24-immunised mice did not produce interleukin (IL)-4, IL-10 or up-regulation of IL-2 mRNA. Cells from rCP24-immunised mice produced IL-10, up-regulated IL-2 mRNA but did not produce IL-4. Neither immunisation with purified CP24 nor injection of pcDNACP24 protected mice against challenge with live smooth B. melitensis. However, the potential of CP24 for a Brucella diagnostic test based on an in vitro antigen (Ag)-specific IFN-gamma production or DTH test would be worth testing.

Animals↗

PubMed2002

Lipopolysaccharide heterogeneity in Brucella strains isolated from marine mammals.

Smooth lipopolysaccharides (S-LPSs) from Brucella strains isolated from seals, dolphins, porpoises, an otter and a minke whale were characterized by ELISA using monoclonal antibodies (mAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes and by Western blot after SDS-PAGE. All strains studied were A-dominant as shown by specific polyclonal sera and this was also confirmed by the mAbs. However, binding patterns in ELISA of mAbs to the specific common (C) epitopes were rather heterogeneous, and for some strains, such as those isolated from striped dolphins, binding of these mAbs was much reduced or negative as had previously been shown for Brucella suis biovar 2 strains. Western blot after SDS-PAGE showed the typical A-dominant strain banding pattern for all marine mammal Brucella isolates, but the average S-LPS size was shorter in many of these compared to reference Brucella abortus strain 544. Thus, S-LPSs of the marine mammal isolates show heterogeneity with regard to their O-PS C epitope content and their average size.

Animals↗

PubMed2002

The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar typhimurium phage type DT204.

Salmonella enterica serovar Typhimurium phage type DT204 strains isolated from cattle and animal feed in Belgium were characterized for high-level fluoroquinolone resistance mechanisms [MICs to enrofloxacin (Enr) and ciprofloxacin (Cip), 64 and 32 microg/ml, respectively]. These strains isolated during the periods 1991-1994, and in 2000 were clonally related as shown by pulsed-field gel electrophoresis (PFGE). Selected strains studied carried several mutations in the quinolone target genes, i.e., a double mutation in the quinolone resistance-determining region (QRDR) of gyrA leading to amino acid changes Ser83Ala and Asp87Asn, a single mutation in the QRDR of gyrB leading to amino acid change Ser464Phe, and a single mutation in the QRDR of parC leading to amino acid change Ser80Ile. Moreover, Western blot analysis showed overproduction of the AcrA periplasmic protein belonging to the AcrAB-ToIC efflux system. This suggested active efflux as additional resistance mechanism resulting in a multiple antibiotic resistance (MAR) phenotype, which was measurable by an increased level of resistance to the structurally unrelated antibiotic florfenicol in the absence of the specific floR resistance gene. The importance of the AcrAB-TolC efflux system in high-level fluoroquinolone resistance was further confirmed by inactivating the acrB gene coding for the multidrug transporter. This resulted in a 32-fold reduction of resistance level to Enr (MIC = 2 microg/ml) and actually in a susceptible phenotype according to clinical breakpoints. Thus, AcrB plays a major role in high-level fluoroquinolone resistance, even when multiple target gene mutations are present. The same effect was obtained using the recently identified efflux pump inhibitor (EPI) Phe-Arg-naphthylamide also termed MC207,110. Among several fluoroquinolones tested in combination with EPI, the MIC of Enr was reduced most significantly. Thus, using EPI together with fluoroquinolones such as Enr may be promising in combination therapy against high-level fluoroquinolone-resistant S. enterica serovar Typhimurium.

Anti-Infective Agents↗

PubMed2002

Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin.

Significant levels of fluoroquinolone resistance were obtained in Campylobacterjejuni isolates after an unique step of selection using enrofloxacin. An Asp90-to-Asn and a Thr86-to-Ile change in the gyrase subunit GyrA were found associated with a low (MIC < or = 8 /microg/ml) or a high (MIC > or = 16 microg/ml) level of resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC > or = 128 microg/ml). Further steps of selection increased the MICs of fluoroquinolones but did not result in a multiple antibiotic resistance phenotype. The Thr86-to-Ile change was found to confer different levels of resistance, pointing out other mechanisms of resistance. However, sequencing revealed no mutation in gyrB, and several attempts did not enable any amplification of the parC gene coding for topoisomerase IV, suggesting an absence of this secondary target in C. jejuni. In addition, no difference in the major outer membrane protein expression was found among the isolates. Furthermore, the use of the recently identified efflux pump inhibitor Phe-Arg-beta-naphthylamide did not result in a significant decrease of fluoroquinolone MICs or change in the frequency of isolation of enrofloxacin-resistant mutants, and thus appears ineffective against fluoroquinolone-resistant C. jejuni isolates. Results obtained during ciprofloxacin accumulation studies confirmed that efflux probably plays a minor role in fluoroquinolone resistance of C. jejuni.

Anti-Infective Agents↗