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Axel Cloeckaert

Publications and source records attributed to Axel Cloeckaert.

At least 37 records · Page 2Linked to original sources
PubMed2005

Resistance to fluoroquinolones linked to gyrA and par C mutations and overexpression of acr AB efflux pump in Salmonella enterica serotype Choleraesuis.

Between 2000 and 2002, 60 clinical isolates of Salmonella enterica serotype Choleraesuis were collected to investigate the mechanism of fluoroquinolone resistance. PCR and sequencing were performed to identify mutations in gyrA, gyrB, par C, the Acr AB-TolC efflux pump regulator, acr R, and the global regulons mar RAB and sox RS. All resistant strains showed mutations in the target genes leading to amino acid changes of Ser 83 Phe and Asp 87 Asn in GyrA and Ser 80 Ile in Par C. A mutation in gyrB was linked to the serotype genetic diversity but not to fluoroquinolone resistance. An efflux pump inhibitor, Phe-Arg-beta-naphthylamide, caused fourfold lower MIC of ciprofloxacin in the resistant isolates, indicating that efflux systems are involved in fluoroquinolone resistance. Western blot analysis showed moderate overproduction of Acr A in fluoroquinolone- resistant isolates. A mutation in acr R gave rise to an internal stop codon in both ciprofloxacin-resistant and -susceptible isolates, suggesting another serotype genetic diversity. No mutations were detected in mar RAB and sox RS among the isolates examined. Cross-resistance to three fluoroquinolones was observed, but gatifloxacin demonstrated relatively lower MICs than those of ciprofloxacin and levofloxacin. Fluoroquinolone resistance in S. Choleraesuis appears to be the combination effect of multiple mutations in various target genes and overexpression of the Acr AB-TolC efflux pump.

Blotting, Western↗
PubMed2005

The Salmonella genomic island 1 is an integrative mobilizable element.

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).

Amino Acid Sequence↗
PubMed2005

Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar typhimurium DT204 acrB mutants selected with fluoroquinolones.

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

Animals↗
PubMed2005

Florfenicol resistance gene floR is part of a novel transposon.

The florfenicol/chloramphenicol resistance gene floR was found to be part of the novel 4,284-bp transposon TnfloR from Escherichia coli. TnfloR consists of the gene floR, a putative regulatory gene, and the transposase gene tnpA. A circular form of TnfloR was detected and suggested the potential mobility of this transposon.

Anti-Bacterial Agents↗
PubMed2005

Structural and functional study of the phenicol-specific efflux pump FloR belonging to the major facilitator superfamily.

The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.

Amino Acid Sequence↗
PubMed2004

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium.

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.

Anti-Bacterial Agents↗
PubMed2004

Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis.

The live attenuated Brucella melitensis Rev.1 strain is considered the best vaccine available for the prophylaxis of brucellosis in sheep caused by either B. melitensis or Brucella ovis. However, its application stimulates antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in infected animals. The periplasmic protein BP26 and the outer membrane protein (OMP) Omp31 are immunodominant antigens in the serological responses of B. melitensis and B. ovis infected sheep, respectively. Accordingly, vaccine strain Rev.1 single and double deletion mutants of the bp26 and omp31 genes were developed, based on the principle that the use of such mutants as vaccines in association with diagnostic tests based on BP26 and Omp31 antigens would allow the serological differentiation between infected and vaccinated animals. The deletion mutants obtained were indistinguishable from the parental Rev.1 strain by conventional bacteriological and typing tests. The expression of their major surface antigens, as determined by reactivity with specific monoclonal antibodies (MAbs), remained unaffected, i.e. smooth-lipopolysaccharide (S-LPS) and OMPs besides in the expression of the antigens whose respective genes were deleted. The bp26 and omp31 deletions did not modify the kinetics of splenic infection nor the residual virulence of Rev.1 in the BALB/c mouse model. Vaccination of BALB/c mice with the deletion mutants conferred significant protective immunity against B. melitensis strain H38 or B. ovis strain PA challenges, to the same extent as that induced by parental Rev.1 strain. Thus, these Rev.1 bp26 or omp31 deletion mutants are promising vaccine candidates against B. melitensis and B. ovis infections and will be further evaluated in sheep.

Animals↗
PubMed2004

Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States.

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.

Anti-Bacterial Agents↗
PubMed2004

Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204.

OBJECTIVES: To study the role of TolC and of parC mutation in high-level fluoroquinolone resistance in clonal clinical strains of Salmonella enterica serotype Typhimurium phage type DT204 (S. Typhimurium DT204). METHODS: Deletion of the tolC gene (DeltatolC) was first performed in a susceptible S. Typhimurium DT104 strain lacking target gene mutations involved in fluoroquinolone resistance. P22 transduction was further used to transduce DeltatolC from this strain to a high-level fluoroquinolone-resistant S. Typhimurium DT204 strain carrying several target gene mutations, including one in parC (ciprofloxacin MIC of 32 mg/L). RESULTS: Deletion of tolC in the high-level fluoroquinolone-resistant S. Typhimurium DT204 strain resulted in the same decrease in resistance levels (16- to 32-fold) as shown previously for an acrB mutant of the same strain, suggesting that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. In some S. Typhimurium DT204 DeltatolC transductants, concomitant loss of the parC (Ser-80-->Ile) mutation, located approximately 9.3 kb upstream of tolC, resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones and thus a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L). CONCLUSION: The AcrAB-TolC efflux system, together with multiple target gene mutations, including the parC mutation, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.

Bacterial Outer Membrane Proteins↗
PubMed2004

Molecular basis of bacterial resistance to chloramphenicol and florfenicol.

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.

Animals↗
PubMed2004

Relative contribution of target gene mutation and efflux to fluoroquinolone and erythromycin resistance, in French poultry and pig isolates of Campylobacter coli.

Thirty-eight avian and swine French isolates of Campylobacter coli were studied for their mechanisms of co-resistance to fluoroquinolones and erythromycin. A Thr86Ile modification of GyrA, responsible for fluoroquinolone resistance, was found in all the strains. Two different levels of resistance to erythromycin (MIC of 8-16 or >/=256 mg/l) were observed. A A2075G mutation in the 23S rRNA genes was found only in the highly-resistant strains. Phe-Arg-beta-naphthylamide, an efflux pump inhibitor, potentiated erythromycin in all the strains examined but restored susceptibility only in the strains with a low-level of resistance. This suggests the involvement of efflux in intrinsic and in acquired low-level of resistance to erythromycin in C. coli.

Amino Acid Substitution↗
PubMed2004

DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide.

Five genes homologous to the well-known omp25 and omp31 genes, that code for two major Brucella spp. outer membrane proteins (OMPs), have been detected in the genome of Brucella melitensis 16M and Brucella suis 1330. In this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical Brucella species reference strains and in representative strains of the recently proposed species Brucella cetaceae and Brucella pinnipediae that classify the Brucella strains isolated in the last years from marine mammals. Although these genes are quite conserved in the genus Brucella, several important differences have been found between species (i) omp31b contains a premature stop codon in B. canis and B. ovis truncating the encoded protein; (ii) the 5' end of omp31b is deleted in the three biovars of B. melitensis which probably prevents synthesis of Omp31b in this species; (iii) only B. melitensis, B. suis and B. neotomae would be able to synthesize the Omp25b protein with the characteristics shared by the Omp25/Omp31 group of proteins (characteristic signal sequence and C-terminal phenylalanine); (iv) a DNA inversion of 1747 bp including omp25b was detected in B. cetaceae strains; (v) a DNA deletion of about 15 kb was detected in all the six B. ovis strains tested. This deletion in B. ovis includes, among other genes, omp25b and wboA, a gene that has been shown to be required for the synthesis of the O-polysaccharide chain of the Brucella spp. smooth lipopolysaccharide. Several features of the DNA region absent from B. ovis suggest that this DNA fragment is a genomic island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. The DNA polymorphism we have found in this work within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by the Brucella species.

Amino Acid Sequence↗
PubMed2004

AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S. enterica serovar Typhimurium DT104 strains.

Bacterial Proteins↗
PubMed2004

Variant Salmonella genomic island 1 antibiotic resistance gene cluster containing a novel 3'-N-aminoglycoside acetyltransferase gene cassette, aac(3)-Id, in Salmonella enterica serovar newport.

Salmonella genomic island 1 (SGI1) harbors an antibiotic resistance gene cluster and was previously identified in the multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, Paratyphi B, and Albany. This antibiotic resistance gene cluster is a complex class 1 integron and most often confers resistance to ampicillin (Ap), chloramphenicol (Cm)/florfenicol (Ff), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracycline (Tc) (ApCmFfSmSpSuTc profile). Recently, variant SGI1 antibiotic resistance gene clusters conferring different antibiotic resistance profiles have been identified in several S. enterica serovars and were classified as SGI1-A to -G. We identified a new variant SGI1 antibiotic resistance gene cluster in two multidrug-resistant S. enterica serovar Newport strains isolated from humans in France. In these strains, the Sm/Sp resistance gene cassette aadA2 inserted at the first attI1 site was replaced by two other aminoglycoside resistance gene cassettes. The first one contains a new resistance gene encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase that confers resistance to gentamicin (Gm) and sisomicin (Sc). This gene has been named aac(3)-Id. The second one harbors the Sm/Sp resistance gene aadA7. This gene cassette replacement in the SGI1 complex integron of serovar Newport strains constitutes a new variant SGI1 antibiotic resistance gene cluster named SGI1-H. The occurrence of SGI1 in different S. enterica serovars, now including serovar Newport, strengthens the hypothesis of horizontal transfer of SGI1.

Acetyltransferases↗
PubMed2004

Salmonella genomic island 1 multidrug resistance gene clusters in Salmonella enterica serovar Agona isolated in Belgium in 1992 to 2002.

Salmonella genomic island 1 (SGI1) harbors a multidrug resistance (MDR) gene cluster which is a complex class 1 integron. Variant SGI1 MDR gene clusters conferring different MDR profiles have also been identified in several Salmonella enterica serovars and classified as SGI1-A to -F. A retrospective study was undertaken to characterize MDR regions from serovar Agona strains harboring SGI1 isolated from poultry in Belgium between 1992 and 2002. A total of 171 serovar Agona strains, displaying resistance to at least one antibiotic, were studied for the presence of SGI1. SGI1 was detected in 94 serovar Agona strains. The most prevalent variant was SGI1-A (85%), which harbors within the SGI1 complex class 1 integron a common region (CR1) containing orf513, a putative transposase gene, adjacent to the dfrA10 trimethoprim resistance gene. A new variant SGI1 named SGI1-G was identified in two strains. It consisted of the pse-1 gene cassette, as in SGI1-B, but with additional insertion of the orf513/dfrA10 region structure. Seven strains displaying the typical SGI1 MDR profile (Ap Cm Ff Sm Sp Su Tc) showed genetic variation at the 3' end of SGI1. These strains harbored the insertion of the CR1 containing orf513 as in SGI1-A, -D, and -G. However, downstream the right end of CR1, they presented different 7.4- to 8.5-kb deletions of the SGI1 3' end that extended to the chromosomal genes yieE and yieF. These results suggest a possible role of CR1 in deletion formation, as has been reported for some insertion sequences. Pulsed-field gel electrophoresis analysis showed that all the serovar Agona SGI1-carrying strains belonged to a single clone. Thus, SGI1 is largely encountered in serovar Agona strains isolated from poultry in Belgium, the most prevalent variant being SGI1-A. SGI1 MDR region undergoes recombinational events resulting in a diversity of MDR gene clusters.

Anti-Bacterial Agents↗
PubMed2004

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France.

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.

Animals↗