An extended PCR-RFLP assay for detection of parC, parE and gyrA mutations in fluoroquinolone-resistant Streptococcus pneumoniae.
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DNA Gyrase↗
Biomedical subjects
Patrice Courvalin
Publications and source records attributed to Patrice Courvalin.
VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis.
Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.
Amino Acid Sequence↗
Heterologous expression of the enterococcal vanA operon in methicillin-resistant Staphylococcus aureus.
Two methicillin- and vancomycin-resistant Staphylococcus aureus strains, MI-VRSA and PA-VRSA, and Enterococcus faecalis DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the vanA operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the d,d-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and E. faecalis DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of S. aureus PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the vanA operon associated with a longer lag phase before growth resumes after induction by vancomycin.
Anti-Bacterial Agents↗
VanE-type vancomycin-resistant Enterococcus faecalis clinical isolates from Australia.
Three distinct Enterococcus faecalis VanE-type isolates-BM4574, BM4575, and BM4576-obtained in Australia were studied. Expression of the resistance genes was constitutive in BM4575, probably due to a 2-bp deletion into the vanSE gene, and inducible in BM4574 and BM4576. Transcription analysis of the vanE operons suggested that the five genes were cotranscribed from an initiation site located 25 bp upstream from the ATG start codon of vanE.
Australia↗
Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system.
The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153-->Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116-->Leu in AdeR at the first residue of the alpha(5) helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.
Acinetobacter baumannii↗
Invasive Streptococcus pneumoniae in France: antimicrobial resistance, serotype, and molecular epidemiology findings from a monthly national study in 2000 to 2002.
A study of 257 French invasive pneumococci isolated between 2000 and 2002 showed high rates of nonsusceptibility to penicillin and macrolides (50%), contrasting with a low frequency of resistance to amoxicillin or levofloxacin (<1%) and tolerance to vancomycin (0%). The genetic homogeneity of some serogroups, including serogroup 1, enhanced the risk of epidemiological changes.
Adult↗
Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR.
A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis and nuc of S. aureus and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant S. aureus, that may escape phenotype-based automated rapid methods.
Anti-Bacterial Agents↗
Glycopeptide antibiotics: from conventional molecules to new derivatives.
Vancomycin and teicoplanin are still the only glycopeptide antibiotics available for use in humans. Emergence of resistance in enterococci and staphylococci has led to restriction of their use to severe infections caused by Gram-positive bacteria for which no other alternative is acceptable (because of resistance or allergy). In parallel, considerable efforts have been made to produce semisynthetic glycopeptides with improved pharmacokinetic and pharmacodynamic properties, and with activity towards resistant strains. Several molecules have now been obtained, helping to better delineate structure-activity relationships. Two are being currently evaluated for skin and soft tissue infections and are in phases II/III. The first, oritavancin (LY333328), is the 4'-chlorobiphenylmethyl derivative of chloroeremomycin, an analogue to vancomycin. It is characterised by: i) a spectrum covering vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and to some extent glycopeptide-intermediate S. aureus (GISA); ii) rapid bactericidal activity including against the intracellular forms of enterococci and staphylococci; and iii) a prolonged half-life, allowing for daily administration. The second molecule is dalbavancin (BI397), a derivative of the teicoplanin analogue A40926. Dalbavancin has a spectrum of activity similar to that of oritavancin against vancomycin-sensitive strains, but is not active against VRE. It can be administered once a week, based on its prolonged retention in the organism. Despite these remarkable properties, the use of these potent agents should be restricted to severe infections, as should the older glycopeptides, with an extension towards resistant or poorly sensitive bacteria, to limit the risk of potential selection of resistance.
Animals↗
Expression of glycopeptide-resistance gene in response to vancomycin and teicoplanin in the cardiac vegetations of rabbits infected with VanB-type Enterococcus faecalis.
VanB-type resistance in enterococci corresponds to resistance to vancomycin but not to resistance to the related glycopeptide teicoplanin, because the vanB gene cluster is activated by the VanR(B)-VanS(B) 2-component regulatory system in response to vancomycin but not to teicoplanin. Mutations in the vanS(B) gene allow for constitutive or teicoplanin-inducible expression of the resistance genes. To analyze in vivo expression of the van genes in rabbits with experimental endocarditis, a VanB-type Enterococcus faecalis with a transcriptional fusion between the P(YB) promoter of resistance genes and the gfpmut1 gene for the green-fluorescent protein in the chromosome was constructed. Rounded heaps containing fluorescent bacteria were detected in vegetation slides from rabbits treated with vancomycin but not in those from control rabbits, revealing induction of a tightly regulated vanB gene cluster. Teicoplanin-resistant mutants were detected as fluorescent bacteria in rabbits treated with teicoplanin. Thus, the reporter system monitored expression of a glycopeptide-resistance gene in vivo at a single-cell level.
Animals↗
Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.
Synercid, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 A resolution in the absence of ligands, at 2.8 A resolution bound to dalfopristin, and at 3.0 A resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >105-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid resistance.
Acetyltransferases↗
VanA-mediated high level glycopeptide resistance in MRSA.
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Bacterial Proteins↗
The vanG glycopeptide resistance operon from Enterococcus faecalis revisited.
Acquired VanG-type resistance to vancomycin (MIC = 16 micro g ml(-1)) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d-alanine-d-serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3' end encoded VanG, a d-Ala:d-Ser ligase, VanXY(G), a putative bifunctional d,d-peptidase and VanT(G), a serine racemase: VanG and VanT(G) were implicated in the synthesis of d-Ala:d-Ser as in VanC- and VanE-type strains. Upstream from the structural genes for these proteins were vanW(G) with unknown function and vanY(G) containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP-MurNAc-tetrapeptide in the cytoplasm. Without the frameshift mutation, VanY(G) had homology with Zn2+ dependent d,d-carboxypeptidases. The 5' end of the gene cluster contained three genes vanU(G), vanR(G) and vanS(G) encoding a putative regulatory system, which were co-transcribed constitutively from the PY(G) promoter, whereas transcription of vanY(G),W(G),G,XY(G),T(G) was inducible and initiated from the P(YG) promoter. Transfer of VanG-type glycopeptide resistance to E. faecalis JH2-2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB-encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element.
3' Flanking Region↗
A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.
Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.
Bacterial Proteins↗
European survey of vancomycin-resistant enterococci in at-risk hospital wards and in vitro susceptibility testing of ramoplanin against these isolates.
A survey in eight European countries, including 13 hospitals, of vancomycin-resistant enterococci (VRE) in at-risk hospital wards (such as the ICU and the haematology ward) was performed in 2001, and the in vitro susceptibility of the isolates ramoplanin and other drugs was tested. A total of 1314 non-duplicate clinical enterococcal isolates were collected, and 38 (2.9%) were vancomycin resistant: 27 Enterococcus faecium and 11 Enterococcus faecalis; 35 VanA and three VanB phenotypes. Rates of VRE among clinical enterococcal isolates varied between 0 and 1.7% for the participating countries, except the UK (10.4%) and Italy (19.6%). One hundred and twenty-three (3.5%) VRE were found among 3499 stool samples tested for the presence of these organisms: 111 (3.2%) E. faecium and 12 (0.3%) E. faecalis; 114 (3.3%) VanA and nine (0.3%) VanB phenotypes. Rates of intestinal colonization with VRE varied between 0 and 1.2% for the participating countries, except Italy (7.5%) and the UK (32.6%). In vitro susceptibility testing showed that the Italian and UK VRE are multi-resistant (including resistance to ampicillin and high-level resistance to gentamicin and streptomycin), and that ramoplanin was active against all strains of VRE, with an MIC90 of 0.5 mg/L for clinical isolates. Pulsed-field gel electrophoresis showed that the high prevalence of VRE in the Italian and UK centres was related to the monoclonal emergence and spread of three centre-specific clones. This survey suggests that in some centres in Europe, a similar situation may be encountered to that in the USA (monoclonal spread of multi-resistant VRE in at-risk wards).
Anti-Bacterial Agents↗
VanD-type vancomycin-resistant Enterococcus faecium 10/96A.
VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.
Amino Acid Sequence↗
Influence of VanD type resistance on activities of glycopeptides in vitro and in experimental endocarditis due to Enterococcus faecium.
The consequences of VanD type glycopeptide resistance on the activity of vancomycin and teicoplanin were evaluated in vitro and in a rabbit model of aortic endocarditis with VanD type clinical isolate Enterococcus faecium BM4339 (MICs: vancomycin, 64 microg/ml; teicoplanin, 4 microg/ml) and its susceptible derivative BM4459 (MICs: vancomycin, 1 microg/ml; teicoplanin, 1 microg/ml). The two antibiotics were inactive against BM4339 in vivo, in terms both of reduction of bacterial counts and of prevention of emergence of glycopeptide-resistant subpopulations, despite using teicoplanin at concentrations greater than the MIC for VanD strains. This could be due to the high inoculum effect also observed in vitro with BM4339 and two other VanD type isolates against both antibiotics. These results suggest that detection of VanD type resistance is of major importance because it abolishes in vivo glycopeptide activity and allows the emergence of mutants highly resistant to glycopeptides.
Animals↗
Efflux pump Lde is associated with fluoroquinolone resistance in Listeria monocytogenes.
Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.
Amino Acid Sequence↗
Chromosomal aadD2 encodes an aminoglycoside nucleotidyltransferase in Bacillus clausii.
Bacillus clausii SIN is one of the four strains of B. clausii composing a probiotic administered to humans for the prevention of gastrointestinal side effects due to oral antibiotic therapy. The strain is resistant to kanamycin, tobramycin, and amikacin. A gene conferring aminoglycoside resistance was cloned into Escherichia coli and sequenced. The gene, called aadD2, encoding a putative 246-amino acid protein, shared 47% identity with ant(4')-Ia from Staphylococcus aureus, which encodes an aminoglycoside 4'-O-nucleotidyltransferase. Phosphocellulose paper-binding assays indicated that the gene product was responsible for nucleotidylation of kanamycin, tobramycin, and amikacin. The aadD2 gene was detected by DNA-DNA hybridization in the three other strains of the probiotic mixture and in the reference strain B. clausii DSM8716, although it did not confer resistance in these strains. Mutations in the sequence of the putative promoter for aadD2 from B. clausii SIN resulted in higher identity with consensus promoter sequences and may account for aminoglycoside resistance in that strain. The aadD2 gene was chromosomally located in all strains and was not transferable by conjugation. These data indicate that chromosomal aadD2 is specific to B. clausii.
Amino Acid Sequence↗