Biomedical subjects

P Courvalin

Publications and source records attributed to P Courvalin.

At least 163 records · Page 9Linked to original sources

PubMed1992

Conjugal transfer of plasmid DNA from Enterococcus faecalis to Escherichia coli in digestive tracts of gnotobiotic mice.

We have studied the transfer of the conjugative shuttle plasmid pAT191, which confers resistance to kanamycin, from Enterococcus faecalis to Escherichia coli in the digestive tracts of six gnotobiotic mice. Colonies of E. coli resistant to kanamycin were isolated from the feces of two mice, respectively, on days 25 and 35 after the beginning of the experiment and never thereafter. The transfer frequency of pAT191, expressed as the number of transconjugants per donor cell isolated from intestines of sacrificed mice, was 3 x 10(-9). These results indicate that conjugation is a mechanism that could account for the resistance gene flux from gram-positive to gram-negative bacteria observed in nature.

Animals↗

PubMed1992

Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci.

The VanA ligase encoded by the vancomycin resistance plasmid pIP816 of Enterococcus faecium BM4147 condenses D-alanine with various D-2-hydroxy and D-2-amino acids in vitro. D-Lactate added to the culture medium restored the vancomycin resistance of a strain that does not produce the VanH dehydrogenase and therefore appears to be a substrate of VanA in vivo.

Drug Resistance, Microbial↗

PubMed1992

Characterization of vanY, a DD-carboxypeptidase from vancomycin-resistant Enterococcus faecium BM4147.

VanY is a protein with a molecular mass of 34.8 kDa encoded by vanY, a member of the high-level vancomycin resistance gene cluster found on plasmid pIP816 in Enterococcus faecium BM4147. Extracts from Escherichia coli JM83 bearing plasmid pAT383, which contains the vanY gene, were examined for enzymatic hydrolysis of peptidoglycan precursors. VanY was associated with the cell membranes and cleaved the C-terminal D-alanine residue of UDP-muramyl-pentapeptide but did not display transpeptidase or beta-lactamase activities. The DD-carboxypeptidase activity was not inhibited by beta-lactam antibiotics. VanY released the C-terminal D-hydroxy acid from depsipeptides produced by the vancomycin resistance protein VanA. These results demonstrate that VanY should contribute in vivo to the hydrolysis of both the D-alanyl-D-alanine- and the depsipeptide-containing peptidoglycan precursors.

Carboxypeptidases↗

PubMed1992

Vancomycin resistance gene vanC is specific to Enterococcus gallinarum.

Nearly all strains of Enterococcus gallinarum are resistant to low levels of vancomycin. The glycopeptide resistance gene vanC from E. gallinarum BM4174 has recently been cloned and sequenced. A probe specific for vanC hybridized with a 2.7-kb EcoRI and a 4.5-kb HindIII fragment of total DNA from the 42 strains of E. gallinarum studied. No homology was detected with DNA of strains belonging to other species intrinsically resistant to vancomycin, including Enterococcus casseliflavus, a species that expresses a vancomycin resistance phenotype similar to that of E. gallinarum. No hybridization with DNA of enterococcal strains with acquired resistance to high or low levels of vancomycin was observed. The specificity of the vanC probe allowed us to distinguish E. gallinarum from 12 other species of enterococci, indicating that this probe is a useful tool for species identification within the genus Enterococcus.

Culture Media↗

PubMed1992

The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR two-component regulatory system encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS two-component regulatory system activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.

Amino Acid Sequence↗

PubMed1991

Characterization of staphylococcal plasmids hybridizing with the fosfomycin resistance gene fosB.

The distribution of the fosB gene, coding for fosfomycin resistance, in 105 fosfomycin-resistant isolates of Staphylococcus from various geographical areas, was studied by Southern blot hybridization. Nucleotide sequences related to fosB were detected in 36 strains belonging to five species. Plasmids bearing fosB were often of a size similar to that of pIP1842 (2.54 kb) in S. epidermidis, most often small (2.4 to 4.1 kb) in other species including S. aureus where a 2.7-kb plasmid was found in 16 out of the 18 strains studied. The fosB gene was geographically dispersed since it was present in six different locations in France and also in Japan. The weak hybridization observed with plasmid DNA of certain strains of S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. warneri may indicate gene heterogeneity for fosfomycin resistance in Staphylococcus spp.

Blotting, Southern↗

PubMed1991

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.

Amino Acid Sequence↗

PubMed1991

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.

Amino Acid Sequence↗

PubMed1991

Mechanisms and implications of glycopeptide resistance in enterococci.

Glycopeptide resistance is recent in enterococci and its expression is inducible by glycopeptides. Two phenotypes can be distinguished: (a) resistance to high levels of vancomycin and teicoplanin, and (b) resistance to low levels of vancomycin only. There is no cross-resistance between glycopeptides, glycolipodepsipeptides (ramoplanin), and lipopeptides (daptomycin). The determinants conferring low-level resistance are nontransferable and presumably chromosomal. High-level resistance is plasmid-mediated and the plasmids range from 34 to 40 kb, are self-transferable, and encode various resistance combinations. All plasmids share the same glycopeptide resistance determinant, which is distinct from that conferring low-level resistance. Induction of resistance is associated with induction of about a 40 kDa protein. We have determined the sequence of the vanA gene encoding one such resistance protein designated VANA. Amino acid sequence similarity was detected between VANA and D-Ala: D-Ala ligases from Enterobacteriaceae. Complementation analysis in Escherichia coli indicated that VANA possesses D-Ala: D-Ala ligase activity and is therefore related to enzymes that catalyze synthesis of glycopeptide target, i.e., terminal D-Ala-D-Ala of peptidoglycan precursors. The contribution of VANA to synthesis of peptidoglycan in the presence of glycopeptides is unknown: VANA could bind to D-Ala-D-Ala, preventing the binding of the drugs; could modify the target of the drug; and could be a ligase with novel specificity.

Anti-Bacterial Agents↗

PubMed1991

Structural relationship between the vancomycin resistance protein VanH and 2-hydroxycarboxylic acid dehydrogenases.

Sequencing upstream from the vanA gene of enterococcal plasmid pIP816 that confers Vm resistance revealed the presence of an ORF which could code for a protein of 322 aa designated VanH. Extensive aa similarity was detected between VanH and 2-hydroxycarboxylic acid dehydrogenase. We discuss possible roles for VanH in the synthesis of a novel type of peptidoglycan precursors with lower affinity for Vm.

Alcohol Oxidoreductases↗

PubMed1991

Sequence of the genes blaT-1B and blaT-2.

We have completed the nucleotide sequence of the genes blaT-1B from transposon Tn2, and blaT-2 from Tn1, which encode the penicillinases TEM-1 and TEM-2, respectively.

Base Sequence↗

PubMed1991

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria.

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.

Cloning, Molecular↗

PubMed1991

Identification of vancomycin resistance protein VanA as a D-alanine:D-alanine ligase of altered substrate specificity.

High-level glycopeptide resistance in Enterococcus faecium BM4147 is mediated by a 38-kDa protein VanA, whose amino acid sequence is related to Gram-negative D-alanine:D-alanine (D-Ala-D-Ala) ligases [Dutka-Malen, S., Molinas, C., Arthur, M., & Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372]. We report purification of VanA and demonstrate that it has D-Ala-D-Ala ligase activity but has substantially modified substrate specificity, compared with Gram-negative D-Ala-D-Ala ligases. VanA preferentially condenses D-Ala with D-Met or D-Phe, raising the possibility that its cellular role is to synthesize a modified cell-wall component, which is subsequently not recognized by vancomycin.

Amino Acid Sequence↗

PubMed1991

Delta-like haemolysin produced by Staphylococcus lugdunensis.

Thirty independent clinical isolates of Staphylococcus lugdunensis were tested for expression of haemolysin characteristics of Staphylococcus aureus and for the presence of DNA sequences with homology to S. aureus toxin genes. Twenty-six strains produced a thermostable haemolysin with greatest activity against rabbit erythrocytes. DNA-DNA hybridisation was performed with S. lugdunesis DNA and probed with the alpha, beta, delta and gamma toxin gene probes of S. aureus as well as that for coagulase. Hybridisation was found with the delta probe in 28 strains including 6 which did not express haemolysin. Two strains did not hybridise with any of the probes. These findings suggest that S. lugdunensis strains produce a delta-like toxin which shares homology with the delta toxin of S. aureus.

Bacterial Proteins↗

PubMed1991

Resistance to trimethoprim and 2,4-diamino-6,7-diisopropyl-pteridine (0/129) in Pasteurella haemolytica.

Thirteen strains of Pasteurella haemolytica resistant to moderate levels of trimethoprim (MICs from 8 to 64 micrograms/ml) and 0/129 (MICs from 16 to 64 micrograms/ml) were isolated from bovine specimens. Two strains, CNP330 and CNP334, were studied and found to harbour various plasmids but all attempts to cure trimethoprim resistance were unsuccessful. Resistance characters were not transferable to Escherichia coli or to Pasteurella multocida by conjugation and to E. coli by transformation. The resistance gene(s) was therefore tentatively assigned to a chromosomal location and cloned into E. coli where it conferred trimethoprim resistance. Trans-complementation analysis of a dihydrofolate reductase-deficient mutant of E. coli showed that trimethoprim resistance was secondary to synthesis of a dihydrofolate reductase. DNA/DNA hybridization of the hybrid plasmid and of strains CNP330 and CNP334 with probes specific for dihydrofolate reductase types I to V were negative, indicating that cross-resistance to trimethoprim and 0/129 in P. haemolytica was due to the acquisition by P. haemolytica of a new resistance determinant.

Animals↗

PubMed1991

Possible in-vivo transfer of beta-lactamase TEM-3 from Klebsiella pneumoniae to Salmonella kedougou.

Salmonella kedougou BM2659 was isolated from the stools and a blood culture of a patient and Klebsiella pneumoniae BM2657 and S. kedougou BM2658 were isolated later from the stools of the same patient. Strains BM2657 and BM2658 had identical resistance phenotypes, to beta-lactams, aminoglycosides and tetracycline, due to the presence of the same genes, blaT, aacA4 and tetC, respectively. Oligotyping indicating that beta-lactam resistance in these strains was encoded by blaT-3 and synthesis of TEM-3 was confirmed by isoelectric focusing. In BM2657 and BM2658, the resistance characters were located on Inc7 or M self-transferable plasmids with indistinguishable EcoRI and HindIII restriction patterns. Southern hybridization of plasmid DNA of these strains with probes pCFFO4, the prototype plasmid encoding TEM-3, genes blaT, aacA4 and tetC gave identical patterns. S. kedougou BM2658 and BM2659 had identical biotypes and serotypes but BM2659 was susceptible to all the study antibiotics. These observations suggest possible transfer, in the digestive tract, of a plasmid encoding TEM-3 beta-lactamase from K. pneumoniae BM2657 to S. kedougou BM2659.

Anti-Bacterial Agents↗

PubMed1991

An IS1-like element is responsible for high-level synthesis of extended-spectrum beta-lactamase TEM-6 in Enterobacteriaceae.

Resistance of Escherichia coli strain HB251 to the newer beta-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum beta-lactamase TEM-6. The corresponding structural gene, bla(T)-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bla(T)-6 differed by two nucleotide substitutions from bla(T)-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bla(T)-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the IS1-like element in clinical isolates of Enterobacteriaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.

Base Sequence↗

PubMed1991

Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice.

Transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes was studied in vitro and in vivo. Tn1545 transferred following filter mating at a frequency of 2.5 x 10(-7) transconjugants per donor colony. A 20-fold increase in transfer frequency was observed when matings were performed in the presence of a subinhibitory concentration of tetracycline. The frequency of in vivo transfer of Tn1545, expressed as the number of transconjugants per donor cell extracted from the intestines of the gnotobiotic mice after 35 days of experiment, was 1.1 x 10(-8). Presence of a low concentration of tetracycline in the drinking water increased this frequency 10-fold.

Animals↗