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PubMed1997

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↗
PubMed1989

Inducible, transferable resistance to vancomycin in Enterococcus faecium, D399.

Enterococcus faecium D399 was isolated from the blood and peritoneal abscess of a patient with intraabdominal sepsis. The patient had not been treated with vancomycin, but the strain was found to be resistant with a MIC of 1000 mg/l. Resistance was inducible and transferable (probably by conjugation) to JH2-2, and correlated with induction of synthesis of a 39 kDa protein. This mechanism appears to be identical to that previously described for E. faecalis A256, suggesting that dissemination of this form of glycopeptide resistance has already occurred. The resistance phenotype of D399, however, differed somewhat from that found in other enterococcal strains with inducible resistance.

Drug Resistance, Microbial↗
PubMedDate not supplied

[Adhesion capacity of Enterococcus faecium (SF 68) and Enterococcus faecalis to various substrates].

The author's studied the adherence ability of Enterococcus faecium SF 68 compared to that of Enterococcus faecalis IM 11f, on various substrates: vascular catheters and cardiac valves of rabbit, immunodepressed rats and diabetic rats. The bacterial adherence test was calculated by the number of bacteria adhered for microscopic field enlarged to 2000. The data obtained by scanning electron microscope SEM (Cambridge Stereoscan 150 MK2) revealed a different adherence action of the two germs on the various substrates. All the adherence tests showed a higher and uniform adherence activity of E. faecalis compared to that of E. faecium.

Animals↗
PubMed2005

Fluorescent in situ hybridization with specific DNA probes offers adequate detection of Enterococcus faecalis and Enterococcus faecium in clinical samples.

Enterococcus faecalis and Enterococcus faecium are among the leading causes of hospital-acquired infections. Reliable and quick identification of E. faecalis and E. faecium is important for accurate treatment and understanding their role in the pathogenesis of infections. Fluorescent in situ hybridization (FISH) of whole bacterial cells with oligonucleotides targeted at the 16S rRNA molecule leads to a reduced time to identification. In clinical practice, FISH therefore can be used in situations in which quick identification is necessary for optimal treatment of the patient. Furthermore, the abundance, spatial distribution and bacterial cell morphology can be observed in situ. This report describes the design of two fluorescent-labelled oligonucleotides that, respectively, detect the 16S rRNA of E. faecalis and the 16S rRNA of E. faecium, Enterococcus hirae, Enterococcus mundtii, Enterococcus villorum and Enterococcus saccharolyticus. Different protocols for the application of these oligonucleotides with FISH in different clinical samples such as faeces or blood cultures are given. Enterococci in a biofilm attached to a biomaterial were also visualized. Embedding of the biomaterial preserved the morphology and therefore the architecture of the biofilm could be observed. The usefulness of other studies describing FISH for detection of enterococci is generally hampered by the fact that they have only focused on one material and one protocol to detect the enterococci. However, the results of this study show that the probes can be used both in the routine laboratory to detect and determine the enterococcal species in different clinical samples and in a research setting to enumerate and detect the enterococci in their physical environment.

Biocompatible Materials↗
PubMed1998

Effect of quinupristin/dalfopristin alone or in combination with vancomycin on the structure of Enterococcus faecium.

Twenty strains of Enterococcus faecium susceptible to quinupristin/dalfopristin (< 2 mg/l) were DNA fingerprinted to exclude strain duplication. Ten strains were susceptible to vancomycin (minimal inhibitory concentration [MIC] < 2 mg/l) and 10 were resistant to vancomycin (MIC > 400 mg/l). Vancomycin at 1/2 MIC, quinupristin/dalfopristin at 1/4 MIC and their combination, except for a tube control, was added to 10 ml trypticase soy broth tubes which were planted with the respective 24-h trypticase soy broth cultures. The products of incubation were sampled periodically throughout 24 h for gram stain and electron microscopy. Cell size was measured on photographs at 20,000x final magnification and results were statistically analyzed. The cells of all strains of Enterococcus faecium exposed for 12 h to quinupristin/dalfopristin were comparable in size to the control, Most cells, however, showed areas of low density of ribosome in the center of the cells. The cells of Enterococcus faecium resistant to vancomycin exposed to vancomycin were larger than the controls with means of 1.96 micron -2.07 micron versus 1.16 micron (p < 0.001); these cells consisted of individual organisms connected by wide cross walls of abnormal fibrous structure. Enterococcus faecium sensitive to vancomycin exposed to vancomycin remained comparable to the control. The combination of quinupristin/dalfopristin plus vancomycin produced large cells with multiple abnormal cross walls in both vancomycin-resistant and vancomycin-sensitive Enterococcus faecium. The addition of quinupristin/dalfopristin to vancomycin appears to modify the vancomycin-susceptible strains to respond to vancomycin in the same manner as do the vancomycin-resistant organisms.

Anti-Bacterial Agents↗
PubMed2006

Characterization of the ends and target site of a novel tetracycline resistance-encoding conjugative transposon from Enterococcus faecium 664.1H1.

Enterococcus faecium 664.1H1 is multiply antibiotic resistant and mercury resistant. In this study, the genetic support for the tetracycline resistance of E. faecium 664.1H1 was characterized. The tet(S) gene is responsible for tetracycline resistance, and this gene is located on the chromosome of E. faecium 664.1H1, on a novel conjugative transposon. The element is transferable to Enterococcus faecalis, where it integrates into a specific site. The element was designated EfcTn1. The integrase of EfcTn1 is related to the integrase proteins found on staphylococcal pathogenicity islands. We show that the transposon is flanked by an 18-bp direct repeat, a copy of which is also present at the target site and at the joint of a circular form, and we propose a mechanism of insertion and excision.

Amino Acid Sequence↗
PubMed2001

Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81.

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called "enterocin 81" [J. Appl. Microbiol. 85 (1998) 521.]. Purification of "enterocin 81" was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C2/C18 reverse-phase chromatographies. The antimicrobial was eluted from the C2/C18 column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4,833.0 and 5,462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.

Amino Acid Sequence↗
PubMed2004

Multiple-locus variable-number tandem repeat analysis, a novel typing scheme to study the genetic relatedness and epidemiology of Enterococcus faecium isolates.

Multiresistant Enterococcus faecium is a major cause of hospital acquired infections and outbreaks. Here, we describe the development of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a novel typing method to assess the genetic relatedness of E. faecium isolates. Six VNTR loci were used to genotype 392 isolates recovered from different animals and human community, hospital survey, and clinical isolates. From 3 to 13 alleles were found per locus, resulting in 127 different MLVA profiles. Clustering of MLVA profiles confirmed the host-specific genogroups found by multilocus sequence typing (MLST) and showed the grouping of clinical and epidemic isolates that belonged to the MLST-C1 cluster in a distinct MLVA-C1 cluster (sensitivity of 97% and specificity of 90%). Furthermore, the discriminatory power of MLVA is comparable to MLST. MLVA profiles appeared to be relatively stable, since isolates from a single outbreak shared the same MLVA profile, which is a prerequisite when MLVA is used to study hospital outbreaks. Our data show that MLVA is a highly reproducible and portable typing method; in contrast to MLST, it is fast, relatively cheap, and easy to perform. Furthermore, it has the abilities of MLST to recognize genetically related and potential epidemic isolates. Submission of MLVA profiles is possible via a Web-based database for international comparison.

Bacterial Typing Techniques↗
PubMed1990

Vancomycin resistance is encoded on a pheromone response plasmid in Enterococcus faecium 228.

In Enterococcus faecium 228, vancomycin resistance is encoded on a 55-kilobase conjugative plasmid, pHKK100. This plasmid was transferred with high frequency into susceptible strains of Enterococcus faecalis and conferred responses to pheromones produced by E. faecalis and Streptococcus sanguis. pHKK100 is the first plasmid described that mediates both vancomycin resistance and pheromone response.

Conjugation, Genetic↗
PubMed2006

Sequence analysis of Enterococcus faecium strain 10/96A (VanD4), the original vancomycin-resistant E. faecium strain in Brazil.

Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil.

Brazil↗
PubMed2006

Bac 32, a novel bacteriocin widely disseminated among clinical isolates of Enterococcus faecium.

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against E. faecium, E. hirae, and E. durans but showed no activity against Listeria monocytogenes. The Bac 32 genetic locus consists of a bacteriocin gene (bacA) and an immunity gene (bacB). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced bacA product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The bacB gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for bacA, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive E. faecium clinical isolates, which suggests that this bacteriocin is widely disseminated among E. faecium strains.

Amino Acid Sequence↗
PubMed1999

Phenotypic characteristics of Enterococcus faecium variants confirmed by intergenic ribosomal polymerase chain reaction and E. faecium polymerase chain reaction.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The emergence of multiple antimicrobial agent-resistant E. faecium has been remarkable; with its strains it is one of the most phenotypically heterogeneous of all enterococcal species. About 15% of enterococcal strains isolated from human clinical specimens were found to have atypical biochemical characteristics. In order to determine if these strains were E. faecium variants, intergenic ribosomal polymerase chain reaction (ITS-PCR) and E. faecium PCR (EfPCR) were performed in 45 atypical strains, and the two PCR results were used to analyze phenotypic characteristics of the strains. As many as 60% (27/45) of the atypical strains were identified as E. faecium. Thus, it is concluded that if an enterococcal strain shows positive reaction to arabinose, arginine, and ribose and negative reaction to methyl-alpha-D-glucopyranoside and pigment, it should be identified as E. faecium.

Bacterial Typing Techniques↗
PubMed1998

Efficient transfer of the pheromone-independent Enterococcus faecium plasmid pMG1 (Gmr) (65.1 kilobases) to Enterococcus strains during broth mating.

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10(-4) per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMbeta1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.

DNA, Bacterial↗
PubMed2002

Identification of vat(E) in Enterococcus faecalis isolates from retail poultry and its transferability to Enterococcus faecium.

Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium.

Acetyltransferases↗
PubMed1993

High-level penicillin resistance and penicillin-gentamicin synergy in Enterococcus faecium.

Thirty-seven Enterococcus faecium strains with different levels of penicillin susceptibility were studied in time-kill experiments with a fixed concentration (5 micrograms/ml) of gentamicin combined with different penicillin concentrations (6 to 600 micrograms/ml). Synergy was defined as a relative decrease in counts of greater than 2 log10 CFU per milliliter after 24 h of incubation when the combination of the antibiotics was compared with its most active component alone. The minimal synergistic penicillin concentrations found were 6 micrograms/ml for 16 of 16 strains for which penicillin MICs were < or = 25 micrograms/ml, 20 to 100 micrograms/ml for 14 of 17 strains for which penicillin MICs were 50 to 200 micrograms/ml, and 200 to 500 micrograms/ml for 4 of 4 strains for which MICs penicillin were > 200 micrograms/ml. Penicillin-gentamicin synergy was observed even in high-level penicillin-resistant E. faecium strains at penicillin concentrations close to one-half the penicillin MIC. The possibility of treating infections caused by high-level penicillin-resistant E. faecium strains with penicillin-gentamicin combinations in particular cases may depend on the penicillin levels attainable in vivo.

Drug Synergism↗
PubMed1995

In vitro activity of RP 59500 (quinupristin/dalfopristin) and ramoplanin against vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREF) collected between July 1991 and February 1994 were tested in vitro against RP 59500 and ramoplanin using agar dilution and standard macro broth dilution procedures. Colony counts were determined at 0, 4, and 24 h. RP 59500 had an MIC range of < or = 0.5-8 micrograms/ml with an MIC90 of 2 micrograms/ml and a MBC range of < or = 0.5-16 micrograms/ml with an MBC90 of 16 micrograms/ml. Ramoplanin had an MIC range of < or = 0.125-1 microgram/ml with an MBC range of < or = 0.125-4 micrograms/ml. The MIC90 for ramoplanin was 1 microgram/ml and the MBC90 was 4 micrograms/ml for the tested isolates. Against these isolates of E. faecium, RP 59500 was bactericidal at 8x MIC, a potentially achievable level using a high drug dosage. Ramoplanin was bactericidal at 2x MIC.

Anti-Bacterial Agents↗
PubMed2001

Aminoglycoside-streptothricin resistance gene cluster aadE-sat4-aphA-3 disseminated among multiresistant isolates of Enterococcus faecium.

Seventy-two Enterococcus faecium isolates of different origins highly resistant to nourseothricin and streptomycin were studied. Sequencing of a genomic fragment from two isolates identified a gene cluster, aadE-sat4-aphA-3, which has been isolated recently in staphylococci and Campylobacter coli. Patterns of digested PCR products of aadE-sat4-aphA-3 were identical for all isolates.

Aminoglycosides↗
PubMed2004

DD1.5k, the gene preferentially expressed in bloodstream isolates of vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREFM) is becoming a threatening pathogen. We identified a gene called DD1.5K by differential display-PCR, which was preferentially expressed in the bloodstream isolates of VREFM. Due to its amino acid similarity to transfer complex protein, trsE, and tissue-specific expression, this gene may be involved in virulence of VREFM.

Adaptation, Physiological↗