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P Courvalin

Publications and source records attributed to P Courvalin.

At least 55 records · Page 3Linked to original sources
PubMed2000

Update on vancomycin resistance.

Enterococci can be responsible for severe infections such as endocarditis, meningitis and septicaemia and are one of the most important causes of nosocomial infections. Resistance in enterococci concerns many classes of antibiotics including, since 1986, glycopeptides. These antibiotics act by blocking cell wall formation, and resistance is due to synthesis of modified peptidoglycan precursors. Resistance can be acquired or intrinsic and strains may be resistant to vancomycin and teicoplanin, or to vancomycin only. Five types of glycopeptide resistance and their biochemical mechanisms have been described. Furthermore, strains that are dependent on vancomycin for growth have been isolated from clinical samples. Data suggest that resistance could originate in glycopeptide-producing organisms or in enterococcal species intrinsically resistant to these antibiotics.

Anti-Bacterial Agents↗
PubMed2000

Modeling of Enterococcus faecalis D-alanine:D-alanine ligase: structure-based study of the active site in the wild-type enzyme and in glycopeptide-dependent mutants.

A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was produced using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template. The model passed critical validation criteria with an accuracy similar to that of the template crystallographic structure and showed that ADP and methylphosphinophosphate were positioned in a large empty pocket at the interface between the central and the C-terminal domains, as in E. coli. It evidenced the residues important for substrate binding and catalytic activity in the active site and demonstrated a large body of conserved interactions between the active sites of the E. faecalis and the E. coli D-Ala:D-Ala ligase, the major differences residing in the balance between the hydrophobic and aromatic environment of the adenine. The model also successfully explained the inactivity of four spontaneous mutants (D295 --> V, which impairs interactions with Mg2+ and R293, which are both essential for binding and catalytic activity; S319 --> I, which perturbs recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in the vicinity of the deletion remains unaltered but phosphate transfer from ATP is perturbed because of lack of K253; T316 --> I, which causes the loss of a hydrogen bond affecting the positioning of S319 and therefore the binding of D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this approach, combining molecular modeling and molecular biology, may help in the design of specific ligands which could inhibit the enzyme and serve as novel antibiotics.

Adenosine Diphosphate↗
PubMed1999

Phylogenetic analysis of proteolytic Acinetobacter strains based on the sequence of genes encoding aminoglycoside 6'-N-acetyltransferases.

The sequence of seven aac(6')-I genes encoding aminoglycoside 6'-N-acetyltransferases from proteolytic Acinetobacter strains including genomic species 14, 15, 16, and 17 and from ungrouped proteolytic strains 631, 640, and BM2722 was determined. Pulsed-field gel electrophoresis of genomic DNA of these strains and of Acinetobacter sp. 6 CIP A165 digested with SfiI followed by hybridization with rRNA and aac(6')-I specific probes indicated that these genes were located in the chromosome. Phylogenetic analysis of the genes indicated that aac(6')-I of A. baumannii, Acinetobacter ungrouped strain 631, and Acinetobacter sp. 16 formed a cluster (91.5 to 92.3% identity) whereas aac(6')-I of Acinetobacter sp. 15, sp. 17, and Acinetobacter ungrouped strain BM2722 formed another cluster (90.7 to 94.6% identity). A third cluster was constituted by A. haemolyticus and Acinetobacter sp. 6 (83.6% identity). The phylogeny drawn from aac(6')-I sequences was consistent with that based on DNA-DNA hybridization and phenotype comparison. The aac(6')-I genes were all species specific except for aac(6')-Ih located in a 13.7-kb non conjugative plasmid from A. baumannii BM2686. We conclude that aac(6')-I genes may be suitable for identification at the species level and for analysis of the phylogenetic relationships of Acinetobacter.

Acetyltransferases↗
PubMed1999

Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

Alanine Racemase↗
PubMed1999

Single-cell analysis of glycopeptide resistance gene expression in teicoplanin-resistant mutants of a VanB-type Enterococcus faecalis.

The vanB gene cluster confers resistance to vancomycin but not to the related antibiotic teicoplanin, as the VanRB SB two-component regulatory system triggers expression of the glycopeptide resistance genes only in response to vancomycin. The VanRB regulator activates promoters PRB and PYB for transcription of the regulatory (vanRB SB) and resistance (vanYB WHB BXB) genes respectively. The gfpmut1 gene encoding a green fluorescent protein was fused to PYB to analyse promoter activation in single cells by fluorescence microscopy and flow cytometry. Characterization of 17 teicoplanin-resistant mutants indicated that amino acid substitutions on either side of the VanSB autophosphorylation site led to a constitutive phenotype. Substitutions in the membrane-associated domain resulted in a gain of function, as they allowed induction by teicoplanin. A vanSB null mutant expressed gfpmut1 at various levels under non-inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of vancomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells rather than from selection of a resistant subpopulation, indicating that VanRB was activated by cross-talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer.

Anti-Bacterial Agents↗
PubMed1999

Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174.

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.

Anti-Bacterial Agents↗
PubMed1999

In-vitro antibacterial activity of levofloxacin against hospital isolates: a multicentre study.

The objective of this study was to evaluate the activity of the fluoroquinolone, levofloxacin, against hospital isolates of bacteria. MICs of levofloxacin were determined for 2154 strains by agar dilution. Breakpoints for susceptibility testing were calculated using the agar diffusion technique with 5 micrograms discs. The activity of levofloxacin against nalidixic acid- and pefloxacin-susceptible Enterobacteriaceae (n = 668) was higher (MIC50/90 0.06-0.12 mg/L) than previously reported for ofloxacin. As seen with other fluoroquinolones, this activity was reduced against nalidixic acid-resistant and pefloxacin-intermediate and -resistant strains (MIC 1-8 mg/L). MICs for Pseudomonas aeruginosa (n = 104) were between 0.12 and 128 mg/L. Levofloxacin had good activity against nalidixic acid- and pefloxacin-susceptible Acinetobacter baumannii (n = 12; MIC 0.06-0.25 mg/L), but the activity was reduced against nalidixic acid- and pefloxacin-resistant strains (n = 80; MIC 1-32 mg/L). Haemophilus influenzae (n = 70), Haemophilus parainfluenzae (n = 47) and Moraxella catarrhalis (n = 64) were inhibited by low concentrations of levofloxacin (MICs 0.016-0.03 mg/L, 0.03-0.12 mg/L) and 0.03-0.12 mg/L, respectively). Clostridium perfringens (n = 23; MIC 0.25-1 mg/L) was more susceptible than Bacteroides fragilis (n = 60; MIC 0.5-4 mg/L). Levofloxacin showed superior activity compared with ofloxacin against methicillin-susceptible staphylococci (n = 107; MIC 0.03-0.5 mg/L); the resistant strains (MICs 2-32 mg/L) were usually also resistant to methicillin. Levofloxacin was less effective against enterococci (n = 105; MIC 1-32 mg/L), but streptococci (n = 192) and pneumococci (n = 129), including 58 penicillin-non-susceptible strains, were inhibited by low concentrations (MICs 0.5-2 mg/L). According to the regression curve, zone diameters were usually 20-22 mm, 17-19 mm and 15-16 mm for MICs of 1, 2 and 4 mg/L, respectively. In conclusion, this study, performed on a large number of strains, confirms the superior anti-bacterial activity of levofloxacin compared with ofloxacin, especially against pathogens isolated from respiratory tract infections.

Acinetobacter↗
PubMed1999

Vancomycin-dependent Enterococcus faecalis clinical isolates and revertant mutants.

Three vancomycin-dependent clinical isolates of Enterococcus faecalis of the VanB type were studied by determining (i) the sequence of the ddl gene encoding the host D-Ala:D-Ala ligase and the vanSB-vanRB genes specifying the two-component regulatory system that activates transcription of the vanB operon, (ii) the level of expression of resistance genes by using DD-dipeptidase activity as a reporter, and (iii) the proportions of the peptidoglycan precursors synthesized. Each strain had a mutation in ddl leading to an amino acid substitution (D295 to V; T316 to I) or deletion (DAK251-253 to E) at invariant positions in D-Ala:D-Ala, D-Ala:D-Lac, and D-Ala:D-Ser ligases. These mutations resulted in impaired host D-Ala:D-Ala ligases since only precursors terminating in D-Ala-D-Lac were synthesized under vancomycin-inducing conditions. Two types of vancomycin-independent revertants of one isolate were obtained in vitro after growth in the absence of vancomycin: (i) vancomycin-resistant, teicoplanin-susceptible mutants had a 6-bp insertion in the host ddl gene, causing the E251-to-EYK change that restored D-Ala:D-Ala ligase activity, (ii) constitutive vancomycin-resistant, teicoplanin-resistant mutants had substitutions (S232 to F or E247 to K) in the vicinity of the autophosphorylation site of the VanSB sensor and produced exclusively precursors ending in D-Ala-D-Lac. Vancomycin- and teicoplanin-dependent mutants obtained by growth in the presence of teicoplanin had an 18-bp deletion in VanSB, affecting residues 402 to 407 and overlapping the G2 ATP binding domain. The rapid emergence of vancomycin-independent revertants in vitro suggests that interruption of vancomycin therapy may not be sufficient to cure patients infected with vancomycin-dependent enterococci.

Anti-Bacterial Agents↗
PubMed1999

Characterization of the chromosomal aac(6')-Iz gene of Stenotrophomonas maltophilia.

The aac(6')-Iz gene of Stenotrophomonas maltophilia BM2690 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 462 bp corresponding to a protein with a calculated mass of 16,506 Da, a value in good agreement with that of ca. 16,000 found by in vitro coupled transcription-translation. Analysis of the deduced amino acid sequence indicated that the protein was a member of the major subfamily of aminoglycoside 6'-N-acetyltransferases. The enzyme conferred resistance to amikacin but not to gentamicin, indicating that it was an AAC(6') of type I. The open reading frame upstream from the aac(6')-Iz gene was homologous to the fprA gene of Myxococcus xanthus (61% identity), which encodes a putative pyridoxine (pyridoxamine) 5'-phosphate oxidase. Pulsed-field gel electrophoresis of total DNA from BM2690 and S. maltophilia ATTC 13637 digested with XbaI, DraI, and SpeI followed by hybridization with rRNA and aac(6')-Iz-specific probes indicated that the gene was located in the chromosome. The aac(6')-Iz gene was detected by DNA-DNA hybridization in all 80 strains of S. maltophilia tested. The MICs of gentamicin against these strains of S. maltophilia were lower than those of amikacin, netilmicin, and tobramycin, indicating that production of AAC(6')-Iz contributes to aminoglycoside resistance in S. maltophilia.

Acetyltransferases↗
PubMed1999

Updated sequence information for TEM beta-lactamase genes.

The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type beta-lactamases have been determined, and an updated blaTEM gene nomenclature is proposed.

DNA, Bacterial↗
PubMed1999

Two-step acquisition of resistance to the teicoplanin-gentamicin combination by VanB-type Enterococcus faecalis in vitro and in experimental endocarditis.

The activity of vancomycin and teicoplanin combined with gentamicin was investigated in vitro against strains of Enterococcus faecalis resistant to vancomycin and susceptible to teicoplanin (VanB type) and against mutants that had acquired resistance to teicoplanin by three different mechanisms. In vitro, gentamicin selected mutants with two- to sixfold increases in the level of resistance to this antibiotic at frequencies of 10(-6) to 10(-7). Teicoplanin selected teicoplanin-resistant mutants at similar frequencies. Both mutations were required to abolish the activity of the gentamicin-teicoplanin combination. As expected, simultaneous acquisition of the two types of mutations was not observed. In therapy with gentamicin or teicoplanin alone, each selected mutants in three of seven rabbits with aortic endocarditis due to VanB-type E. faecalis BM4275. The vancomycin-gentamicin combination selected mutants that were resistant to gentamicin and to the combination. In contrast, the teicoplanin-gentamicin regimen prevented the emergence of mutants resistant to one or both components of the combination. These results suggest that two mutations are also required to suppress the in vivo activity of the teicoplanin-gentamicin combination.

Animals↗
PubMed1999

Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases.

The sequences of the blaTEM genes encoding TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum beta-lactamases were determined. Analysis of the deduced amino acid sequences indicated that TEM-20 and TEM-29 were derived from TEM-1 and that TEM-21 and TEM-22 were derived from TEM-2. The substitutions involved were Ser-238 and Thr-182 for TEM-20; His-164 for TEM-29; Lys-104, Arg-153, and Ser-238 for TEM-21; and Lys-104, Gly-237, and Ser-238 for TEM-22. The promoter region of the blaTEM-22 gene was identical to that of blaTEM-3. High-level production of TEM-20 could result from a 135-bp deletion which combined the -35 region of the Pa promoter with the -10 region of the P3 promoter and a G-->T transition in the latter motif.

Base Sequence↗