Resistance to glycopeptides in enterococci.
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Animals↗
Biomedical subjects
P Courvalin
Publications and source records attributed to P Courvalin.
Selection of glycopeptide-resistant mutants of VanB-type Enterococcus faecalis BM4281 in vitro and in experimental endocarditis.
Enterococcus faecalis BM4281 is resistant to vancomycin, susceptible to teicoplanin (VanB phenotype), and intrinsically resistant to low levels of gentamicin. The efficacy of glycopeptides against BM4281 was investigated in a rabbit model of experimental endocarditis for reduction of bacterial counts in cardiac vegetations and selection of mutants with increased resistance to glycopeptides. Teicoplanin led to a 100-fold reduction of bacteria in the vegetations, whereas vancomycin had no effect. Monotherapy with either antibiotic selected mutants with homogeneous or heterogeneous resistance to high levels of both glycopeptides. Vancomycin also selected mutants that required the antibiotic for growth. The combination of gentamicin plus teicoplanin was bactericidal, prevented the emergence of mutants, and allowed sterilization of the vegetations in 25% of the rabbits, indicating that the combination may be an alternative if penicillin cannot be used against VanB-type enterococci.
Animals↗
Loss of intrinsic aminoglycoside resistance in Acinetobacter haemolyticus as a result of three distinct types of alterations in the aac(6')-Ig gene, including insertion of IS17.
The distribution of the aac(6')-Ig gene, encoding aminoglycoside 6'-N-acetyltransferase-Ig [AAC(6')-Ig], was studied in 96 Acinetobacter haemolyticus strains and 12 proteolytic Acinetobacter strains, including Acinetobacter genomospecies 6, 13, and 14 and 3 unnamed species assigned to this genomic group by DNA-DNA hybridization. This gene was detected by DNA-DNA hybridization in all 96 A. haemolyticus strains and by PCR in 95 strains but was not detected in strains of other species, indicating that it may be used to identify A. haemolyticus. Three A. haemolyticus strains were susceptible to tobramycin and did not produce an aminoglycoside 6'-N-acetylating activity, although they contained aac(6')-Ig-related sequences. An analysis of three susceptible A. haemolyticus strains indicated that aminoglycoside resistance was abolished by the following three distinct mechanisms: (i) a point mutation in aac(6')-Ig that led to a Met56-->Arg substitution, which was shown by analysis of a revertant to be responsible for the loss of resistance; (ii) a polythymine insertion that altered the reading frame; and (iii) insertion of IS17, a new member of the IS903 family. These observations indicated that AAC(6')-Ig is not essential for the viability of A. haemolyticus, although the aac(6')-Ig gene was detected in all members of this species.
Acetyltransferases↗
Active efflux of antimicrobial agents in wild-type strains of enterococci.
Enterococci are intrinsically resistant to numerous antimicrobial agents. We examined the energy-dependent efflux of radiolabeled drugs from four reference strains of Enterococcus faecalis and a strain of Enterococcus faecium and found that most strains pumped out norfloxacin and chloramphenicol. Efflux of tetracycline was detected only in certain strains.
Anti-Bacterial Agents↗
Emergence of the trimethoprim resistance gene dfrD in Listeria monocytogenes BM4293.
The sequence of the trimethoprim resistance gene of the 3.7-kb plasmid (pIP823) that confers high-level resistance (MIC, 1,024 microg/ml) to Listeria monocytogenes BM4293 was determined. The gene was identical to dfrD recently detected in Staphylococcus haemolyticus MUR313. The corresponding protein, S2DHFR, represents the second class of high-level trimethoprim-resistant dihydrofolate reductase identified in gram-positive bacteria. We propose that trimethoprim resistance in L. monocytogenes BM4293 could originate in staphylococci.
Amino Acid Sequence↗
Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae.
We report a mutation in the parE genes of two in vitro mutants of Streptococcus pneumoniae responsible for low-level resistance to fluoroquinolones. Sequential acquisition of mutations in parE and gyrA leads to higher levels of resistance. This confirms that topoisomerase IV is the primary target of fluoroquinolones in S. pneumoniae.
Anti-Infective Agents↗
VanD-type glycopeptide-resistant Enterococcus faecium BM4339.
Enterococcus faecium BM4339 was constitutively resistant to vancomycin (MIC, 64 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml). A 605-bp product obtained with the V1 and V2 primers for amplification of genes encoding D-Ala:D-Ala ligases and related glycopeptide resistance proteins was sequenced after cloning. The deduced amino acid sequence had 69% identity with VanA and VanB and 43% identity with VanC, consistent with the finding that BM4339 synthesized peptidoglycan precursors terminating in D-lactate. This new type of glycopeptide resistance phenotype was designated VanD.
Amino Acid Sequence↗
The VanS sensor negatively controls VanR-mediated transcriptional activation of glycopeptide resistance genes of Tn1546 and related elements in the absence of induction.
Transposon Tn1546 from Enterococcus faecium BM4147 encodes a histidine protein kinase (VanS) and a response regulator (VanR) that regulate transcription of the vanHAX operon encoding a dehydrogenase (VanH), a ligase (VanA), and a D,D-dipeptidase (VanX). These last three enzymes confer resistance to glycopeptide antibiotics by production of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate. Transcription of vanS and the role of VanS in the regulation of the vanHAX operon were analyzed by inserting a cat reporter gene into vanS. Transcription of cat and vanX was inducible by glycopeptides in partial diploids harboring vanS and vanS(omega)cat but was constitutive in strains containing only vanS(omega)cat. Promoters P(R) and P(H), located upstream from vanR and vanH, respectively, were cloned into a promoter probing vector to study transactivation by chromosomally encoded VanR and VanS. The promoters were inactive in the absence of vanR and vanS, inducible by glycopeptides in the presence of both genes, and constitutively activated by VanR in the absence of VanS. Thus, induction of the vanHAX operon involves an amplification loop resulting from binding of phospho-VanR to the P(R) promoter and increased transcription of the vanR and vanS genes. Full activation of P(R) and P(H) by VanR was observed in the absence of VanS, indicating that the sensor negatively controls VanR in the absence of glycopeptides, presumably by dephosphorylation. Activation of the VanR response regulator in the absence of VanS may involve autophosphorylation of VanR with acetyl phosphate or phosphorylation by a heterologous histidine protein kinase.
Anti-Bacterial Agents↗
Identification of clinically relevant viridans group streptococci to the species level by PCR.
A PCR assay that allows identification of clinically relevant viridans group streptococci (Streptococcus gordonii, S. mitis, S. mutans, S. oralis, S. salivarius, and S. sanguis) to the species level and identification of milleri group streptococci (S. anginosus, S. constellatus, and S. intermedius) to the group level was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases which are species specific and ubiquitous in prokaryotes possessing peptidoglycan. The specificity of this assay was tested on 9 reference strains and 91 characterized clinical isolates. This assay offers a specific and rapid alternative to phenotypic or DNA-DNA hybridization methods for identification of clinically relevant viridans group streptococci.
Alanine↗
Epidemiological survey of Neisseria meningitidis susceptibility to penicillin G in France.
The susceptibility of 82 strains of Neisseria meningitidis to penicillin G and amoxicillin was evaluated with two media "gonococci-meningococci" medium (G medium) derived from Mueller Hinton and chocolate agar. Among these 82 strains 52 were isolated from CSF and/or blood and 30 from miscellaneous isolates. G medium was compared with chocolate agar using the correlation between diameters and MIC and the E-test. Routinely standardised antibiogram is still used but the authors added the following techniques 1) MIC of penicillin G is tested (0.06; 0.125; 0.250; 0.50 mg/l); 2) use of oxacillin disc charged with 5 micrograms. Penicillinase producing meningococci were not found. Standardised antibiogram on 4192 strains resulted in a modal distribution of diameters between 18 to 40 mm. Moderate meningococci susceptible strains to penicillin G are increasing in France: 1994: 4%; 1995: 11%; 1996: 18%.
Amoxicillin↗
Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281.
A 64-kb genetic element harboring a vanB vancomycin-resistance (VmR) gene cluster was shown to translocate from the chromosome of Enterococcus faecalis BM4281 into the hemolysin (Hly) plasmid, pIP964. Sequence analysis of the resulting junction fragments indicated that the VmR genes were carried by a composite transposon, Tn1547, bounded by two distantly related insertion sequences (IS), designated IS256-like and IS16, in a direct orientation. IS256-like (1324 bp) was identical to IS256, except for two nucleotide (nt) transitions in the putative transposase gene. IS16 (1466 bp) contained a large open reading frame (ORF) that was 61% identical to the gene encoding the putative transposase of IS256. There was 58% identity between the deduced amino acid (aa) sequences of the putative transposases of IS256-like (390 aa) and IS16 (395 aa). IS16 was delineated by imperfect inverted repeats (IR) (18 out of 26 bp) which were related (23/26 bp identity) to their respective imperfect IR counterparts in IS256. The difference between the IS was not a barrier for transposition of Tn1547 which generated an 8-bp duplication at the target site. Dissemination of VanB-type resistance among enterococci results from two mechanisms: (i) large conjugative elements translocate from chromosome to chromosome following inter-strain transfer; and (ii) as described in this report, the VmR genes transpose from replicon to replicon within the same strain as part of composite transposons that are internal to the conjugative elements.
Amino Acid Sequence↗
Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes.
The D-alanine:D-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesize D-alanyl-D-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesize D-alanyl-D-lactate or d-alanyl-d-serine. The sequence of internal fragments of eight structural d-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (D-alanine:D-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and DdlA and DdlB from enteric bacteria and Haemophilus influenzae constituted separate clusters.
Amino Acid Sequence↗
Glycopeptide resistance in enterococci.
Glycopeptide resistance in enterococci results from the production of peptidoglycan precursors with low affinity for these antibiotics. The mobility of the resistance genes by transposition and conjugation and the ability of the resistance proteins to interfere with synthesis of normal precursors in different hosts indicate that dissemination into other bacterial species should be anticipated.
Anti-Bacterial Agents↗
Mechanisms of glycopeptide resistance in enterococci.
Inducible resistance to high levels of glycopeptide antibiotics in clinical isolates of enterococci is mediated by Tn1546 or related transposons. Tn1546 encodes the VanH dehydrogenase which reduces pyruvate to D-lactate (D-Lac) and the VanA ligase which catalyses synthesis of the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). The depsipeptide replaces the dipeptide D-Ala-D-Ala leading to production of peptidoglycan precursors which bind glycopeptides with reduced affinity. In addition, Tn1546 encodes the VanX dipeptidase and the VanY D,D-carboxypeptidase that hydrolyse the dipeptide D-Ala-D-Ala and the C-terminal D-Ala residue of the cytoplasmic precursor UDP-MurNAC-L-Ala-gamma-D- Glu-L-Lys-D-Ala-D-Ala, respectively. These two proteins act in series to eliminate D-Ala-D-Ala-containing precursors. VanX is required for resistance whereas VanY only slightly increases the level of resistance mediated by VanH, VanA and VanX.
Amino Acid Sequence↗
Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-resistant enterococci.
Transposon Tn1546 from Enterococcus faecium BM4147 mediates high-level resistance to the glycopeptide antibiotics vancomycin and teicoplanin. Tn 1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D-alanyl-D-lactate (D-Ala-D-Lac), a D,D-dipeptidase (VanX) that hydrolyses D-Ala-D-Ala and a two-component regulatory system (VanR-VanS) that controls transcription of the vanHAX operon. Strains of Enterococcus faecalis harbouring various copy numbers of the vanRSHAX cluster were tested to determine if there was a correlation between the levels of resistance to glycopeptides, the levels of expression of the corresponding resistance genes and the relative proportions of the different cytoplasmic peptidoglycan precursors. Increased transcription of the vanHAX operon was associated with increased incorporation of D-Ala-D-Lac into peptidoglycan precursors to the detriment of D-Ala-D-Ala, and with a gradual increase in the vancomycin-resistance levels. More complete elimination of D-Ala-D-Ala-containing precursors was required for teicoplanin resistance. The VanY and VanZ proteins also encoded by Tn1546 were not effectors of the regulation of the vanHAX operon but contributed to vancomycin and teicoplanin resistance, respectively. Differences at the regulatory level accounted for phenotypic diversity in acquired glycopeptide resistance by production of D-lac-ending precursors.
Anti-Bacterial Agents↗
Genetics of glycopeptide resistance in enterococci.
Glycopeptide resistance in enterococci is phenotypically and genotypically heterogeneous. The genes responsible for inducible resistance to high levels of vancomycin and teicoplanin (VanA phenotype) are carried by the 10,851-bp Tn1546 transposon. Transposition of Tn1546 into self-transferable plasmids and subsequent transfer by conjugation appears to be responsible for the dissemination of this type of resistance. Nine polypeptides are encoded by Tn1546 that belong to five functional groups: transposition functions (ORF1 and ORF2), regulation of resistance gene expression (VanR and VanS), synthesis of depsipeptide D-Ala-D-lactate (VanH and VanA), hydrolysis of D-Ala-D-Ala-containing peptidoglycan precursors (VanX and VanY), and low-level teicoplanin resistance (VanZ). VanB-type resistance (various levels of resistance to vancomycin and susceptibility to teicoplanin) is also due to production of D-Ala-D-Lac. The VanB ligase of VanB-type strains is structurally and functionally similar to VanA. The vanB gene was found on composite transposon Tn1547, which, in turn, was part of larger conjugative chromosomally located elements (90 to 250 kb). In contrast to acquired VanA- and VanB-type resistance, VanC-type resistance (low level of resistance to vancomycin and susceptibility to teicoplanin) is an intrinsic property of motile enterococci. Resistance in these species is due to synthesis of dipeptide D-Ala-D-Ser by VanC ligases leading to production of cell wall precursors with reduced vancomycin affinity.
Anti-Bacterial Agents↗
The Garrod Lecture. Evasion of antibiotic action by bacteria.
Antibiotics have reduced the mortality from infectious diseases but not the prevalence of these diseases. Use, and often abuse, of antimicrobial agents encourages the evolution of bacteria toward resistance, often resulting in therapeutic failure. This evolution is due to the emergence of "new' resistance mechanisms and to the spread of well-characterized mechanisms of resistance to the majority of bacterial species. Bacterial resistance can be intrinsic or acquired. Intrinsic resistance is species or genus specific and delineates the spectrum of activity of the antibiotic. Acquired resistance is present in only certain strains of a species or of a genus. The latter results from mutation in a gene located in the host chromosome or a plasmid or from acquisition of new genetic information by a bacterium, mainly by conjugation or transformation. In this review, recent developments in the understanding of biochemical mechanisms and the genetics of resistance is considered for the clinically important antibiotic families.
Anti-Bacterial Agents↗
Efficacy of vancomycin and teicoplanin alone and in combination with streptomycin in experimental, low-level vancomycin-resistant, VanB-type Enterococcus faecalis endocarditis.
The efficacy of vancomycin (VM) and teicoplanin (TE), alone and in combination with streptomycin (SM), against enterococci that express low-level VanB-type VM resistance was investigated in experimental endocarditis using isogenic strains of Enterococcus faecalis susceptible to glycopeptides and aminoglycosides or inducibly resistant to low levels of VM (MIC = 16 micrograms/ml). VM was significantly less active against the resistant strain than against the susceptible strain, establishing that low-level VanB-type VM resistance can influence therapeutic efficacy. By contrast, TE had equally good activity against both strains. VM or TE combined with SM was synergistic and bactericidal against the resistant strain in vitro. While both combinations were efficient in reducing bacterial density in vivo, TE plus SM was significantly superior to VM plus SM if valve sterilization was considered. These data suggest that despite the presence of low-level VanB-type resistance, combination therapy with a glycopeptide and SM (and presumably other aminoglycosides to which there is not high-level resistance) will nevertheless provide effective bactericidal activity.
Animals↗