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P Courvalin

Publications and source records attributed to P Courvalin.

At least 109 records · Page 6Linked to original sources
PubMed1996

Specificity of induction of glycopeptide resistance genes in Enterococcus faecalis.

Regulation of VanA- and VanB-type glycopeptide resistance in enterococci is mediated by related two-component regulatory systems (VanR-VanS and VanRB-VanSB). The transglycosylase inhibitors vancomycin, teicoplanin, and moenomycin induced synthesis of the VanX D,D-dipeptidase in a VanA-type Enterococcus faecalis harboring transposon Tn1546. Inhibitors of reactions immediately preceding (ramoplanin) or following (penicillin G and bacitracin) transglycosylation were not inducers. These results identify accumulation of membrane-bound lipid intermediate II as a potential signal for induction of VanA-type resistance. In E.faecalis BM4281 harboring a wild vanB genetic element, D,D-dipeptidase synthesis was only inducible by vancomycin. Induction of the production of the VanB ligase by vancomycin was required for growth of a vancomycin-dependent derivative of BM4281, since introduction of a plasmid coding for constitutive synthesis of the VanA ligase eliminated the requirement of glycopeptide for growth. Both vancomycin and teicoplanin were able to induce D,D-dipeptidase synthesis in BM4281 derivatives that were vancomycin and teicoplanin resistant or vancomycin and teicoplanin dependent. Acquisition of teicoplanin resistance in the latter types of strains was due to alteration in induction specificity associated with an increase in the sensitivity of the regulatory system to vancomycin. Thus, the wild VanRB-VanSB system is unable or not sensitive enough to sense teicoplanin, although mutations can lead to recognition of this antibiotic.

Anti-Bacterial Agents↗
PubMed1996

Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro.

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.

Amino Acid Sequence↗
PubMed1996

Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene.

We have analyzed the development of fluoroquinolone resistance between 1986 and 1993 among clinical isolates of Enterococcus faecalis from a French hospital. One hundred randomly selected isolates per year were screened for resistance to ciprofloxacin (MIC > 2 micrograms/ml) and for high-level resistance to gentamicin (MIC > 1,000 micrograms/ml). The percentages of ciprofloxacin-resistant strains for these years were as follows: 1986, 0; 1987, 1; 1988 to 1989, 2; 1990, 6; 1991, 16; 1992, 24; and 1993, 14. Eighty-three percent of the ciprofloxacin-resistant isolates were coresistant to high levels of gentamicin. Forty-eight high-level gentamicin-resistant E. faecalis strains, which were resistant (24 strains) or susceptible (24 strains) to ciprofloxacin, were examined by pulsed-field gel electrophoresis (PFGE) of SmaI-digested total DNA. Numerous PFGE types were observed among the ciprofloxacin-susceptible isolates, whereas one type was largely predominant among the ciprofloxacin-resistant strains, which suggests that the increase in fluoroquinolone resistance was due to the spread of a single clone. A 241-bp fragment of gyrA, corresponding to the quinolone resistance-determining region, was amplified and sequenced for seven ciprofloxacin-resistant isolates. Six strains had high levels of resistance (MICs, 32 to 64 micrograms/ml) and had a mutation at position 83 (Escherichia coli coordinates) from Ser to Arg (three strains) or to Ile (two strains) or at position 87 from Glu to Gly (one strain), whereas the low-level-resistant isolate (MIC, 8 micrograms/ml) had no mutations.

Anti-Infective Agents↗
PubMed1996

Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro.

The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.

Animals↗
PubMed1996

Regulation of VanB-type vancomycin resistance gene expression by the VanS(B)-VanR (B) two-component regulatory system in Enterococcus faecalis V583.

Acquired VanA- and VanB-type glycopeptide resistance in enterococci is due to synthesis of modified peptidoglycan precursors terminating in D-lactate. As opposed to VanA-type strains which are resistant to both vancomycin and teicoplanin, VanB-type strains remain teicoplanin susceptible. We have determined the sequence of a 7,160-bp DNA fragment associated with VanB-type resistance in Enterococcus faecalis V583 that contains seven open reading frames. The distal part encoded the VanH (B), VanB, and VanX (B) proteins that are highly similar to the putative VanH, VanA, and VanX proteins responsible for VanA-type resistance. Upstream from the structural genes for these proteins were the vanY(B) gene encoding a D,D-carboxypeptidase and an open reading frame vanW with an unknown function. The proximal part of the gene cluster coded for the apparent VanS(B)-VanR (B) two-component regulatory system. VanR (B) was related to response regulators of the OmpR subclass, and VanS (B) was related to membrane-associated histidine protein kinases. Analysis of transcriptional fusions with a reporter gene and promoter mapping indicated that the VanR B-VanS B two-component regulatory system activates a promoter located immediately downstream from the vanS B gene. Vancomycin, but not teicoplanin, was an inducer, which explains teicoplanin susceptibility of VanB-type enterococci.

Amino Acid Sequence↗
PubMed1995

The vanZ gene of Tn1546 from Enterococcus faecium BM4147 confers resistance to teicoplanin.

A five-gene cluster from Tn1546 confers resistance to the glycopeptide antibiotics vancomycin (Vm) and teicoplanin (Te) by synthesis of pentadepsipeptide peptidoglycan precursors terminating in D-lactate, which replaces D-alanine in the same position of precursors utilized by susceptible enterococci. Cloning and nucleotide sequencing indicated that Tn1546 contains an additional gene, designated vanZ, which confers low-level Te resistance, in the absence of the genes required for pentadepsipeptide synthesis. Analysis of cytoplasmic peptidoglycan precursors, accumulated in the presence of ramoplanin, showed that VanZ-mediated Te resistance does not involve incorporation of a substituent of D-alanine into the precursors.

Amino Acid Sequence↗
PubMed1995

Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp.

Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.

Amino Acid Sequence↗
PubMed1995

Stability of TEM beta-lactamase mutants hydrolyzing third generation cephalosporins.

The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179. The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.

Arginine↗
PubMed1995

Incidence of antibiotic resistance in Listeria species.

To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.

Anti-Bacterial Agents↗
PubMed1995

Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR.

A PCR assay that allows simultaneous detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci (Enterococcus faecium, E. faecalis, E. gallinarum, and E. casseliflavus) was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance proteins. The specificity of the assay was tested on 5 well-characterized glycopeptide-resistant strains and on 15 susceptible enterococcal type strains. Clinical isolates of enterococci that could not be identified to the species level by conventional methods were identified by the PCR test. This assay offers a specific and rapid alternative to antibiotic susceptibility tests, in particular for detection of low-level vancomycin resistance.

Anti-Bacterial Agents↗
PubMed1995

Gene transfer from bacteria to mammalian cells.

Transfer of genetic information between phylogenetically remote bacterial genera [1], from bacteria to yeast [2] and from bacteria to plants [3] by plasmid conjugation has been described. However, direct DNA transfer from prokaryotes to mammalian cells has not yet been demonstrated. Certain bacterial species have evolved the ability to enter mammalian cells by inducing their own internalization [4]. We show that invasive strains of Shigella flexneri and Escherichia coli, that undergo lysis upon entry into mammalian cells because of impaired cell wall synthesis, can act as stable DNA delivery systems to their host. This direct gene transfer is efficient, of broad host cell range and the replicative or integrative vectors so delivered are stably inherited and expressed by the cell progeny. DNA delivery by abortive invasion of eukaryotic cells by bacteria is of potential interest for stimulation of mucosal immunity and for in vivo or ex vivo gene therapy of human diseases.

Animals↗
PubMed1994

TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis.

The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins. On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes. Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations. Analysis of the double mutants proved more hazardous. Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

Aztreonam↗
PubMed1994

Characterization of the aac(6')-Ik gene of Acinetobacter sp. 6.

The aac(6')-Ik gene which confers resistance to aminoglycosides in Acinetobacter sp. 6 CIP A165 was characterized. The resistance gene was identified as a coding sequence of 438 bases pairs corresponding to a protein with a calculated mass of 16627 Da. Alignment of aac(6')-Ik with aac(6')-Ig from Acinetobacter haemolyticus indicated 83% identity. Like aac(6')-Ig of A. haemolyticus and aac(6')-Ij of Acinetobacter sp. 13, the aac(6')-Ik gene was apparently located in the chromosome and was species specific. The high degree of identity between aac(6')-Ig and -Ik, compared with genomic DNA relationships between the host species, indicated that these genes have diverged from a common ancestor in a parental Acinetobacter species.

Acetyltransferases↗
PubMed1994

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583.

Acquired resistance to glycopeptides in enterococci is associated with the production of D-Alanine:D-Alanine ligase-related proteins. The VanA protein associated with high-level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D-alanine-D-lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over-production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.

Amino Acid Sequence↗
PubMed1994

Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174.

Vancomycin resistance in enterococci is an increasing clinical problem, and several phenotypes have been identified. We demonstrate here that the resistance mechanism in the constitutively vancomycin-resistant Enterococcus gallinarum BM4174 involves an altered pathway of peptidoglycan synthesis and hydrolysis of the normal precursors in the vancomycin-sensitive pathway. A ligase encoded by the vanC gene catalyses synthesis of D-Ala-D-Ser and substitutes this dipeptide for D-Ala-D-Ala in peptidoglycan precursors. It is presumed that this substitution lowers the affinity of vancomycin for its target site. Destruction of D-Ala-D-Ala (D,D-peptidase activity) and of UDP-MurNAc-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala by removal of the terminal D-Ala residue (D,D-carboxypeptidase activity) ensures that the normal vancomycin-sensitive pathway of peptidoglycan synthesis cannot function in the resistant strain.

Amino Acid Sequence↗