Moderate-level resistance to glycopeptide LY333328 mediated by genes of the vanA and vanB clusters in enterococci.

Three of five natural plasmids carrying a wild-type vanA gene cluster did not confer LY333328 glycopeptide resistance on Enterococcus faecalis JH2-2 (MIC = 2 microg/ml). The two remaining plasmids conferred resistance to the drug (MIC, 8 microg/ml). The vanB gene cluster did not confer resistance to LY333328, since this antibiotic was not an inducer. Mutations in the vanS(B) sensor gene that allowed induction by teicoplanin or constitutive expression of the vanB cluster led to LY333328 resistance (MIC, 8 to 16 microg/ml). Overproduction of the VanH, VanA, and VanX proteins for D-alanyl-D-lactate (D-Ala-D-Lac) synthesis and D-Ala-D-Ala hydrolysis was sufficient for resistance to LY333328 (MIC, 16 microg/ml). Mutations in the host D-Ala:D-Ala ligase contributed to LY333328 resistance in certain VanA- and VanB-type strains, but the MICs of the antibiotic did not exceed 16 microg/ml. Addition of D-2-hydroxybutyrate in the culture medium of mutants that did not produce the VanH D-lactate dehydrogenase led to incorporation of this D-2-hydroxy acid at the C-terminal ends of the peptidoglycan precursors and to LY333328 resistance (MIC, 64 microg/ml). The vanZ gene of the vanA cluster conferred resistance to LY333328 (MIC, 8 microg/ml) by an unknown mechanism. These data indicate that VanA- and VanB-type enterococci may acquire moderate-level resistance to LY333328 (MIC 相似文献   

Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil

The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.     

Inducible, transferable resistance to vancomycin in Enterococcus faecalis A256.

A strain of Enterococcus faecalis (A256) was isolated from the urine of a patient with urinary sepsis and was found to exhibit susceptibilities (micrograms per milliliter) to various glycopeptides as follows: vancomycin, 256; teicoplanin, 16; 62208, 512; 62211, 4; and 62476, 16. As judged by growth rates before and after exposure to sub-MICs of glycopeptides, vancomycin and 62476 induced self-resistance, 62208 and 62211 induced slight self-resistance, and teicoplanin did not induce self-resistance. Vancomycin induced cross-resistance to all other glycopeptides tested, as judged both in growth experiments and by direct measurement of inhibition of peptidoglycan synthesis in cells exposed to sub-MICs of vancomycin. Thus, the spectra of activity of the glycopeptides were not correlated with their patterns of induction. There was a correlation between the increased synthesis of a 39-kilodalton (kDa) protein located in the cytoplasmic membrane and the induction of resistance. Protoplasts of A256 were susceptible to inhibition of peptidoglycan synthesis by vancomycin at levels similar to those for susceptible strains. Vancomycin resistance was transferable on filters from the parent strain to E. faecalis JH2-2 at a frequency of about 10(-7), and the 39-kDa protein was also inducible by glycopeptides in these transconjugants. We conclude that A256 is resistant to glycopeptides by virtue of the synthesis of a 39-kDa cytoplasmic membrane protein, that this protein is probably involved in preventing access of the glycopeptides to their peptidoglycan targets, and that this resistance is transferable, probably by conjugation.     

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.     

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.     

Low-level resistance to glycopeptides amongst staphylococcus species: surveillance in a university hospital and evaluation of a vancomycin screening agar.

The prevalence of low-level resistance to glycopeptides (teicoplanin MIC > or = 8 microg/mL and vancomycin MIC > or = 4 microg/mL) among staphylococci was investigated over a 15 month period. A total of 2,279 isolates (1,519 S. aureus, 760 coagulase-negative staphylococcus (CNS)) were screened using inoculum of 10(6) CFU/mL and Mueller-Hinton agars supplemented with 8 microg/mL of teicoplanin. Of these, 218 isolates (136 S. aureus and 82 CNS) grew on the screening agar. For these isolates, teicoplanin and vancomycin MICs were determined by agar dilution method and a vancomycin agar screening method was evaluated. The prevalence of low-level resistance to teicoplanin and vancomycin was 7.8% and 0.1% for S. aureus and 8.8% and 0.8% for CNS, respectively. The brain heart infusion agar containing 4 microg/mL of vancomycin failed to detect two out of eight staphylococcal isolates with vancomycin MICs of 4 microg/mL. Furthermore, the method appeared to lack reproducibility. Considering the increasing incidence of vancomycin treatment failure in staphylococcal infection, a more reliable screening method is required.     

Glycopeptide-resistant Enterococcus faecium BM4416 is a VanD-type strain with an impaired D-Alanine:D-Alanine ligase

VanD-type Enterococcus faecium BM4416 was constitutively resistant to vancomycin and to teicoplanin by synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate. Like E. faecium BM4339, the only VanD-type strain described so far, BM4416 produced an impaired D-alanine:D-alanine ligase. Unlike for BM4339, which had a 5-bp insertion in ddl, inactivation of the gene in BM4416 was due to insertion of IS19.     

Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin

Many VISA (vancomycin intermediately resistant Staphylococcus aureus) strains are characterized by increased cell wall biosynthesis and decreased cross-linking of the peptide side chains, leading to accumulation of free D-alanyl-D-alanine termini in the peptidoglycan, which act as false target sites for vancomycin. A spontaneous mutant of methicillin-resistant VISA strain SA137/93A (vancomycin MIC [E-test], 8 micro g/ml), called SA137/93G, showed increased resistance to vancomycin (MIC [E-test], 12 micro g/ml). Analysis of the resistance profile of the mutant revealed a loss of beta-lactam resistance with a concomitant increase in resistance to glycopeptides. In both strains, cell wall thickness was 1.4-fold greater than that of control isolates. However, cross-linking of the cell wall was drastically lower in SA137/93A than in SA137/93G. The sensitivity of strain SA137/93G to beta-lactams was due to loss of the beta-lactamase plasmid and a deletion that comprises 32.5 kb of the methicillin resistance cassette SCCmec, as well as 65.4 kb of chromosomal DNA. A spontaneous mutant of SA137/93G with higher sensitivity to vancomycin displayed a cell wall profile similar, in some respects, to that of an fmhB mutant. Results described here and elsewhere show that the only feature common to all VISA strains is a thickened cell wall, which may play a central role in the vancomycin resistance mechanism.     

Glycopeptide susceptibility profiles of Staphylococcus haemolyticus bloodstream isolates

Twelve clinical strains of Staphylococcus haemolyticus (eight methicillin resistant and three methicillin susceptible), isolated from blood cultures between 1982 and 1997, were investigated for teicoplanin and vancomycin susceptibility profiles. On the basis of conventional MIC tests and breakpoints, four isolates were susceptible (MICs, 1 to 8 microgram/ml) and eight were resistant (MICs, 32 to 64 microgram/ml) to teicoplanin while all were susceptible to vancomycin (MICs, 1 to 2 microgram/ml). All four strains for which the conventional teicoplanin MICs were within the range of susceptibility expressed heterogeneous resistance to teicoplanin and homogeneous vancomycin susceptibility. Of the eight strains for which the conventional teicoplanin MICs were within the range of resistance, six expressed heterogeneous and two expressed homogeneous teicoplanin resistance while seven showed heterogeneous vancomycin resistance profiles (with subpopulations growing on 8 microgram of the drug per ml at frequencies of >/=10(-6) for six strains and 10(-7) for one) and one demonstrated homogeneous vancomycin susceptibility. Of six bloodstream isolates of other staphylococcal species (S. aureus, S. epidermidis, and S. simulans), for all of which the conventional teicoplanin MICs were >/=4 microgram/ml and the vancomycin MICs were 相似文献   

Rapid depletion of free vancomycin in medium in the presence of beta-lactam antibiotics and growth restoration in Staphylococcus aureus strains with beta-lactam-induced vancomycin resistance

A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of beta-lactam antibiotics (beta-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the D-Ala-D-Ala terminal of the peptidoglycan network is replaced with D-Ala-D-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of approximately 0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 microg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of beta-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the beta-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 microg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below approximately 1.5 microg/ml at 24 h, the cells began to grow. In the culture supplemented with the beta-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 microg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of approximately 1.7 microg/ml or at 4 h of incubation. The gene encoding the enzyme involved in D-Ala-D-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by beta-lactam antibiotics.     

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.     

D-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium.     

VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405.

Enterococcus faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 microg/ml) and was susceptible to teicoplanin (MIC, 0.5 microg/ml). No PCR product was obtained when the total DNA of this clinical isolate was used as a template with primers specific for glycopeptide resistance genes vanA, vanB, vanC, and vanD. However, a 604-bp PCR fragment was obtained when V1 and V2 degenerate primers were used and total DNA was digested with HindIII as a template. The product was cloned and sequenced. The deduced amino acid sequence had greater identity (55%) with VanC than with VanA (45%), VanB (43%), or VanD (44%). This was consistent with the fact that BM4405 synthesized peptidoglycan precursors that terminated in D-serine residues. After induction with vancomycin, weak D,D-dipeptidase and penicillin-insensitive D,D-carboxypeptidase activities were detected in cytoplasmic extracts of BM4405, whereas a serine racemase activity was found in the membrane preparation. This new type of acquired glycopeptide resistance was named VanE.     

Antimicrobial activity of MDL 62,873, a semisynthetic derivative of teicoplanin, in vitro and in experimental infections.

MDL 62,873 is an amide derivative of teicoplanin A2-2. Like those of natural glycopeptides, its antibacterial activity is mediated by inhibition of cell wall peptidoglycan synthesis. Against streptococci and enterococci, the in vitro activity of MDL 62,873 was similar to that of teicoplanin and greater than that of vancomycin. Against staphylococci, it has activity similar to that of vancomycin, and it was significantly more active than teicoplanin against coagulase-negative isolates. Like teicoplanin and vancomycin, MDL 62,873 had slow but significant bactericidal activity (99 to 99.9% killing in 24 h) against staphylococci at concentrations near the MIC. In murine septicemia studies with Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, the 50% effective doses were lower than those of vancomycin. In staphylococcal endocarditis in rats, MDL 62,873 at 20 mg/kg of body weight and vancomycin at 40 mg/kg, both doses given intravenously twice daily, had similar efficacies in reducing the heart bacterial load. These results probably reflect the longer half-life of MDL 62,873, which has a pharmacokinetic profile in rats similar to that of teicoplanin.     

Bacteremia due to Staphylococcus aureus with reduced susceptibility to vancomycin

Four cases of bacteremia caused by Staphylococcus aureus with heteroresistance to vancomycin (hetero-VRSA) were described. In at least two of these four mortalities, the cause of death was temporally related to the hetero-VRSA bacteremia. The vancomycin and teicoplanin MICs of the resistant subpopulations of these four hetero-VRSA were 8 and 24 microg/ml, respectively. All isolates were producers of beta-lactamase, produced penicillin-binding protein PBP2a, and possessed the mecA gene accounting for methicillin resistance. Thickening of the peptidoglycan cell wall was observed by electron microscopy. When ampicillin was combined with vancomycin, in vitro synergism was detected using the checkerboard titration method (epsilonFIC = 0.13). The use of vancomycin plus ampicillin-sulbactam could be a viable option in treating severe hetero-VRSA infection in view of the higher affinity of ampicillin toward PBP2a.     

Genetic characterization of vanG, a novel vancomycin resistance locus of Enterococcus faecalis

Enterococcus faecalis strain WCH9 displays a moderate level of resistance to vancomycin (MIC = 16 microgram/ml) and full susceptibility to teicoplanin but is negative by PCR analysis using primers specific for all known enterococcal vancomycin resistance genotypes (vanA, vanB, vanC, vanD, and vanE). We have isolated and sequenced a novel putative vancomycin resistance locus (designated vanG), which contains seven open reading frames, from this strain. These are organized differently from those of all the other enterococcal van loci, and, furthermore, the individual vanG gene products exhibit less than 50% amino acid sequence identity to other van gene products.     

Phenotypic and genotypic heterogeneity of glycopeptide resistance determinants in gram-positive bacteria

Gram-positive glycopeptide-resistant bacteria isolated in various hospitals in Europe and in the United States between 1986 and 1988 were collected. Three resistance phenotypes could be distinguished. Thirty-one enterococci were highly resistant to vancomycin and teicoplanin. Resistance was transferable to other enterococci by conjugation for 16 of the 22 isolates that were tested. Homology was detected by hybridization between a probe specific for the vanA gene, which encodes an inducible high-level glycopeptide resistance protein in Enterococcus faecium BM4147, and DNA of the 31 clinical isolates and the 16 corresponding transconjugants. This indicates that a single class of resistance determinants accounts for high-level glycopeptide resistance in enterococci. The strains differed in their biotypes and resistance phenotypes and harbored resistance plasmids of various sizes, suggesting that spread of this resistance phenotype is due to dissemination of a gene rather than of a bacterial clone or of a single plasmid. Four enterococcal isolates were resistant to low levels of vancomycin and susceptible to teicoplanin. Twenty-three coagulase-negative staphylococcal isolates were resistant to teicoplanin and susceptible to vancomycin. These two groups of strains did not hybridize with the vanA probe and did not transfer resistance at a detectable frequency. The vanA gene was not detected in the glycopeptide-producing strains of Amycolatopsis orientalis (vancomycin) and Actinoplanes teichomyceticus (teicoplanin) or in various gram-positive bacteria intrinsically resistant to glycopeptides.     

Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus.

The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor.     

Consequences of VanE-type resistance on efficacy of glycopeptides in vitro and in experimental endocarditis due to Enterococcus faecalis

The consequences on glycopeptide activity of low-level resistance to vancomycin due to VanE-type resistance were evaluated in vitro and in experimental endocarditis caused by Enterococcus faecalis BM4405 (MICs of vancomycin and teicoplanin: 16 and 0.5 microg/ml, respectively), its susceptible derivative BM4405-1, and susceptible E. faecalis JH2-2. After 24 h of incubation, vancomycin at 8 microg/ml was not active against E. faecalis BM4405 whereas it was bacteriostatic against strains BM4405-1 and JH2-2. Against all three strains, vancomycin at 30 microg/ml and teicoplanin at 8 or 30 microg/ml were bacteriostatic but bactericidal when combined with gentamicin. In rabbits with aortic endocarditis due to VanE-type resistant strain BM4405, treatment with a standard dose of vancomycin generated subinhibitory plasma concentrations (i.e., peak of 36.3 +/- 2.1 microg/ml and trough of 6.0 +/- 2.2 microg/ml) and led to no significant reduction in mean aortic valve vegetation counts compared to no treatment of control animals. In contrast, a higher dosing regimen of vancomycin (i.e., resulting in a peak of 38.3 +/- 5.2 microg/ml and a trough of 15.0 +/- 8.3 microg/ml), providing plasma concentrations above the MIC for the entire dosing interval, led to significant and similar activities against all three strains, which were enhanced by combination with gentamicin. Treatment with teicoplanin led to results similar to those obtained with vancomycin at a high dose. No subpopulations with increased resistance to glycopeptides were selected in vitro or in vivo. In conclusion, the use of a high dose of vancomycin was necessary for the treatment of experimental enterococcal endocarditis due to VanE-type strains.