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Rapid detection and differentiation method of VanA, VanB and VanC phenotypes in vancomycin-resistant enterococci.

We developed a simple method that can replace the polymerase chain reaction (PCR) to distinguish between vancomycin-resistant enterococci (VRE) with the vanA, vanB and vanC genes. The method is based on induction of teicoplanin resistance by vancomycin in vanB-VRE, while the two compounds have a synergistic effect in vanC-VRE. In addition, vanA-VRE shows resistance to both vancomycin and teicoplanin, and both the compounds can induce resistance to vanA-VRE. Utilising these properties, we attempted to develop a simple method to distinguish between vanA, vanB and vanC. We compared our simple method with the PCR method in 43 strains of vanA-VRE, 35 strains of vanB-VRE and 37 strains of vanC-VRE. The results were 100% consistent with that obtained by PCR.

Anti-Bacterial Agents↗

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins.

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.

Animals↗

Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall.

Glycopeptide antibiotics, including vancomycin, form complexes via a set of five hydrogen bonds with the acyl-l-Lys-d-Ala-d-Ala portion of the peptidyl stems of the bacterial cell wall peptidoglycan. This complexation deprives the organism from the ability to cross-link peptidyl stems of the peptidoglycan, leading to bacterial cell death. Four synthetic fragments as surrogates of the components of the bacterial cell wall have been prepared in our lab in multistep syntheses. These synthetic samples were used in investigations of the thermodynamics properties (DeltaG degrees , DeltaH degrees , and TDeltaS degrees ) for the complexation with vancomycin by isothermal titration calorimetry (ITC). Complexation with the glycopeptide analogues is largely enthalpy-driven (formation of five hydrogen bonds), and in the analogues with a single peptidyl stem, the complexation is 1:1. The complexation is more complicated with an approximately 2 kDa cell wall surrogate (compound 4), which possesses two peptidyl stems. The data were suggestive of interactions between the two vancomycin molecules, with an entropic penalty attributable to restriction of molecular movements within the complex due to restriction of motion of the highly mobile acyl-d-Ala-d-Ala moiety of the peptidyl stems. These data were reconciled with the recently determined NMR solution structure for the peptidoglycan fragment 4 and its implications for the larger cell wall.

Anti-Bacterial Agents↗

Antibacterial activity of calozeyloxanthone isolated from Calophyllum species against vancomycin-resistant Enterococci (VRE) and synergism with antibiotics.

Calozeyloxanthone ( 1) was re-isolated from the root bark of Calophyllum moonii, an endemic species of Sri Lanka, and found to be active against vancomycin-resistant Enterococci (VRE) and vancomycin-sensitive Enterococci (VSE) with MIC values of 6.25 microg/ml and 12.5 microg/ml, respectively. Further, a marked synergism between 1 and vancomycin hydrochloride (VCM) against VRE was also observed. These findings suggest that 1 in combination with VCM against VRE may be useful in controlling VRE infections.

Anti-Bacterial Agents↗

Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate.

Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of Escherichia coli penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit E. coli PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.

Bacterial Proteins↗

A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology.

VanD-mediated glycopeptide resistance has been reported for an isolate of Enterococcus faecium, BM4339. Three clinical isolates of vancomycin-resistant E. faecium collected from 3 patients during a 6-week period in 1993 had agar dilution MICs of vancomycin and teicoplanin of 128 and 4 microg/mL, respectively. Polymerase chain reaction (PCR) using degenerate primers complementary to genes encoding d-Ala-d-X ligases yielded a 630-bp product that was similar to the published partial sequence of vanD. By use of inverse PCR, vanD, vanHD, and two partial flanking open-reading frames were sequenced. The deduced amino acid sequence of VanD showed 67% identity with VanA and VanB. vanD appeared to be located on the chromosome and was not transferable to other enterococci. The 3 isolates were indistinguishable by pulsed-field gel electrophoresis and differed from BM4339. No other isolates carrying vanD were found in a subset of 875 recent US isolates of vancomycin-resistant enterococci.

Amino Acid Sequence↗

Vancomycin-intermediate Staphylococcus aureus with phenotypic susceptibility to methicillin in a patient with recurrent bacteremia.

Vancomycin-intermediate Staphylococcus aureus (VISA) are an emerging problem. We observed a statistically significant inverse relationship in the MICs of vancomycin and oxacillin in S. aureus isolates from a patient undergoing hemodialysis who received 26 weeks of treatment with vancomycin during November 1999 through April 2000. All isolates were mecA positive and were indistinguishable by pulsed-field gel electrophoresis. The evolving susceptibility patterns of this strain highlight the challenges of detecting and treating VISA infections.

Bacteremia↗

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↗

Frequency of reduced vancomycin susceptibility and heterogeneous subpopulation in persistent or recurrent methicillin-resistant Staphylococcus aureus bacteremia.

Vancomycin susceptibility was checked in isolates from initial and final blood samples obtained from 22 patients with persistent or recurrent methicillin-resistant Staphylococcus aureus bacteremia. The minimum inhibitory concentration of vancomycin was determined using Etest and found to have increased in 2 pairs of isolates, and results of screening in 4 mu g vancomycin and a modified population analysis profile suggested heteroresistance in 3 isolates (13.6%). Heteroresistance is not a common cause of persistent or recurrent bacteremia.

Anti-Bacterial Agents↗

Practical guidelines for vancomycin usage, with prospective drug-utilization evaluation.

To strengthen guidelines for vancomycin use, practical guidelines were developed. A prospective survey was conducted of all patients receiving vancomycin during two 1-month periods, 1 year apart, during which significant improvements were noted. Practical guidelines may contribute to appropriateness of vancomycin use, serve as educational tools, and facilitate improved surveillance.

Drug Utilization Review↗

Risk factors for vancomycin-resistant Enterococcus (VRE) infection in colonized patients with cancer.

To determine the risk factors for vancomycin-resistant Enterococcus (VRE) infection in colonized patients with cancer, we conducted a case-control study. According to multivariate analysis, the only significant factors were neutropenia (< 500 cells/mm3) for more than 1 week and the use of oral vancomycin. Therefore, colonized neutropenic patients with cancer who have previously used oral vancomycin are most prone to VRE infection.

Adolescent↗

Reduction of vancomycin-resistant enterococcal infections by limitation of broad-spectrum cephalosporin use in a trauma and burn intensive care unit.

Both vancomycin and third-generation cephalosporin use are believed to contribute to a rise in vancomycin-resistant enterococci (VRE) infections. In 1998, the largest number of VRE infections in our hospital occurred in the trauma/burn intensive care unit (TBICU), accounting for nearly 20% of hospital infections. In an attempt to control the VRE infection rate, antibiotic protocols for prophylaxis, empiric, and definitive therapy were initiated during the final quarter of 1998 to minimize cephalosporin use by the introduction of piperacillin/tazobactam. Therefore, we undertook a study of the VRE infection rate for the TBICU in relation to vancomycin, piperacillin/tazobactam, piperacillin, third-generation cephalosporin, and total cephalosporin use before and after efforts to limit cephalosporins. These data were compared to those in the medical and surgical intensive care units. During 1998, seven VRE infections occurred in the TBICU. Following initiation of antibiotic protocols, one case of VRE infection occurred in the subsequent month and no cases in the 17 months since. The decrease in the VRE infection rate corresponded with a significant increase in the use of piperacillin/tazobactam and a reduction in third-generation and total cephalosporin use. In contrast, cephalosporin use in the medical and surgical intensive care units remains significantly higher than in the TBICU, and neither unit has had a reduction in their VRE infection rates.

Burn Units↗

Antimicrobial susceptibility testing of vancomycin-resistant Enterococcus by the VITEK 2 system, and comparison with two NCCLS reference methods.

We evaluated the automated VITEK 2 system (bioMerieux) for antimicrobial susceptibility testing of vancomycin-resistant Enterococcus (VRE). The results obtained with the VITEK 2 system were compared to those obtained using two NCCLS reference methods. The VITEK 2 system produced MICs for penicillin G, erythromycin and vancomycin that were very similar to those of the reference agar-dilution test with all results being within a twofold dilution. When MICs of teicoplanin for these isolates were measured by the agar-dilution method and VITEK 2 system, there was one 'very major' error and seven 'minor' errors. There were no 'major' errors for any of the antibiotics tested. When the results obtained by the micro broth-dilution method were compared with those obtained by the VITEK 2 system, there was one 'very major' error for teicoplanin by the VITEK 2 system, as was the case with the agar-dilution method. There were two 'minor' errors for erythromycin and seven 'minor' errors for teicoplanin. There were no 'major' errors for any of the antibiotics tested. The 35 VRE strains identified phenotypically by the VITEK 2 Advanced Expert System included nine of Enterococcus faecalis and 23 of Enterococcus faecium. Neither Enterococcus avium nor Enterococcus hirae were identified. A total of 32 phenotypes were classified into 22 VanA and 10 VanB strains. PCR genotyping demonstrated 23 vanA+ and nine vanB+ strains. There were differences between the VITEK 2 system results and those of PCR. Overall, 54.3 % of the test results were obtained within 7 h. All MIC values for the 35 VRE isolates were determined within 13 h of completing incubation. The VITEK 2 system is a simple method for accurately detecting vancomycin-resistant strains of Enterococcus and can be used to rapidly determine MICs.

Anti-Bacterial Agents↗

Emergence of VanD-type vancomycin-resistant Enterococcus faecium in Stockholm, Sweden.

A vancomycin-resistant Enterococcus faecium isolate from the urine of a liver transplant patient in Stockholm was found to contain a vanD gene. The sequence of the vanD PCR product shared 100% identity with the vanD5 allele. The isolate was resistant to a relatively high level of vancomycin (128 mg/L) and a low level of teicoplanin (4 mg/L). This is the first VanD-type vancomycin-resistant E. faecium isolate reported in Sweden. The emergence of this strain reinforces the necessity of infection control efforts to interrupt the spread of these organisms.

Alleles↗

Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus.

Vancomycin is usually reserved for treatment of serious infections, including those caused by multidrug-resistant Staphylococcus aureus. A clinical isolate of S. aureus with high-level resistance to vancomycin (minimal inhibitory concentration = 1024 microg/ml) was isolated in June 2002. This isolate harbored a 57.9-kilobase multiresistance conjugative plasmid within which Tn1546 (vanA) was integrated. Additional elements on the plasmid encoded resistance to trimethoprim (dfrA), beta-lactams (blaZ), aminoglycosides (aacA-aphD), and disinfectants (qacC). Genetic analyses suggest that the long-anticipated transfer of vancomycin resistance to a methicillin-resistant S. aureus occurred in vivo by interspecies transfer of Tn1546 from a co-isolate of Enterococcus faecalis.

Anti-Bacterial Agents↗

Nitrofurantoin is active against vancomycin-resistant enterococci.

The activity of nitrofurantoin was tested against 300 isolates of Enterococcus faecium, Enterococcus faecalis, and Enterococcus gallinarum. No isolates tested were resistant to nitrofurantoin (MIC, >/=128 microg/ml), including vancomycin-resistant E. faecium isolates with vanA- and vanB-positive genotypes and vancomycin-resistant E. gallinarum isolates. We conclude that nitrofurantoin may provide effective treatment of urinary tract infections caused by vancomycin-resistant enterococci.

Anti-Bacterial Agents↗

Occurrence, structure, and mobility of Tn1546-like elements in environmental isolates of vancomycin-resistant enterococci.

The occurrence, structure, and mobility of Tn1546-like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored vanA. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as Enterococcus faecium (n = 13), E. casseliflavus (n = 6), E. mundtii (n = 3), E. faecalis (n = 3), and E. durans (n = 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn1546-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn1546 in the prototype strain E. faecium BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for E. casseliflavus strains. Indistinguishable Tn1546-like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to E. faecium BM4105 was demonstrated for E. faecalis, E. mundtii, and E. durans. This study indicates that VRE, including species other than E. faecium and E. faecalis, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn1546-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.

Animals↗

Heteroresistance to vancomycin in Enterococcus faecium.

This study presents the first report of vancomycin heteroresistance in an Enterococcus faecium isolate from a patient. The original isolate was susceptible in vitro to vancomycin. E-tests showed growth of subcolonies in a zone of inhibition with a vancomycin MIC of >256 microg/ml. Both the susceptible and resistant colonies were from the same strain as determined by PFGE, and both contained the vanA gene as determined by PCR.

Anti-Bacterial Agents↗